U.S. patent application number 12/158262 was filed with the patent office on 2009-07-16 for genemap of the human genes associated with crohn's disease.
Invention is credited to Rene Allard, Abdelmajid Belouchi, Walter Edward Bradley, Sandie Briand, Pascal Croteau, Helene Fournier, Tim Keith, Randall David Little, Quynh Nguyen-Huu, Bruno Paquin, John Verner Raelson, Jonathan Segal, Paul Van Eerdewegh.
Application Number | 20090181380 12/158262 |
Document ID | / |
Family ID | 38189115 |
Filed Date | 2009-07-16 |
United States Patent
Application |
20090181380 |
Kind Code |
A1 |
Belouchi; Abdelmajid ; et
al. |
July 16, 2009 |
GENEMAP OF THE HUMAN GENES ASSOCIATED WITH CROHN'S DISEASE
Abstract
The present invention relates to the selection of a set of
polymorphism makers for use in genome wide association studies
based on linkage disequilibrium mapping. In particular, the
invention relates to the fields of pharmacogenomics, diagnostics,
patient therapy and the use of genetic haplotype information to
predict an individual's susceptibility to IBD (ex: Chrohn's
disease) and/or their response to a particular drug or drugs.
Inventors: |
Belouchi; Abdelmajid; (Ville
St.-Laurent, CA) ; Raelson; John Verner; (Hodson,
CA) ; Bradley; Walter Edward; (Montreal, CA) ;
Paquin; Bruno; (Chateangay, CA) ; Fournier;
Helene; (Montreal, CA) ; Nguyen-Huu; Quynh;
(Longveuil, CA) ; Croteau; Pascal; (Laval, CA)
; Allard; Rene; (Montreal, CA) ; Briand;
Sandie; (Montreal, CA) ; Van Eerdewegh; Paul;
(Carlisle, MA) ; Little; Randall David; (Laval,
CA) ; Segal; Jonathan; (Efrat, IL) ; Keith;
Tim; (Bedford, MA) |
Correspondence
Address: |
Ralph A. Dowell of DOWELL & DOWELL P.C.
2111 Eisenhower Ave, Suite 406
Alexandria
VA
22314
US
|
Family ID: |
38189115 |
Appl. No.: |
12/158262 |
Filed: |
December 19, 2006 |
PCT Filed: |
December 19, 2006 |
PCT NO: |
PCT/US06/48246 |
371 Date: |
November 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60751420 |
Dec 19, 2005 |
|
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Current U.S.
Class: |
435/6.11 ;
436/501; 702/19; 703/11 |
Current CPC
Class: |
C12Q 2600/158 20130101;
Y02A 90/26 20180101; Y02A 90/10 20180101; C12Q 2600/172 20130101;
C12Q 1/6883 20130101; C12Q 2600/156 20130101 |
Class at
Publication: |
435/6 ; 703/11;
436/501; 702/19 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G06F 19/00 20060101 G06F019/00; G01N 33/53 20060101
G01N033/53 |
Claims
1. A method of constructing a GeneMap for Crohn's disease
comprising identifying at least two chromosomal loci associated
with Crohn's disease in a population, wherein said at least two
chromosomal loci are selected from the genomic regions listed in
Table 1.
2.-20. (canceled)
21. A method of diagnosing Crohn's disease, the predisposition to
Crohn's disease, or the progression of Crohn's disease in an
individual, comprising comparing (i) the amount and/or
concentration of at least one polypeptide encoded in Candidate
Region 341 and/or at least one nucleic acid encoding the
polypeptide in a biological sample from the individual with (ii) a
control amount and/or concentration of the at least one polypeptide
and/or the at least one nucleic acid; wherein a significant
difference between the amount and/or concentration of the at least
one polypeptide and/or the at least one nucleic acid of (i) and the
control amount and/or concentration of the at least one polypeptide
and/or the at least one nucleic acid of (ii) is indicative of a
Crohn's disease, the predisposition to Cronh's disease or the
progression of Crohn's disease in the individual.
22. (canceled)
23. The method of claim 21, wherein the amount and/or concentration
of the at least one nucleic acid is determined with a nucleic acid
probe.
24. The method of claim 23, wherein said nucleic acid probe is
selected from group consisting of SEQ ID NOs: 3747, 3749, 3751,
3753, 3755, 3757, 3759; 3761, 3763, 3765, 3767, 3771, 3773, 3775,
3777, 3779, 3781, 3783, 3785, 3787, 3789, 3791, 3793, 3795, 3797,
3799, 3801, 3803, 3805, 3807, 3809, 3811, 3813, 3815, 3817, 3819,
3821, 3823, 3825, 3827, 3829, 3831, 3833, 3835, 3837, 3839, 3841,
3843, 3845, 3847, 3849, 3851, 3853, 3855, 3857, 3859, 3861, 3863,
3865 3887 and 3889 or a fragment thereof.
25. The method of claim 23, wherein said nucleic acid probe is
capable of hybridizing to a sequences selected from group
consisting of SEQ ID NOs: 3747, 3749, 3751, 3753, 3755, 3757, 3759;
3761, 3763, 3765, 3767, 3771, 3773, 3775, 3777, 3779, 3781, 3783,
3785, 3787, 3789, 3791, 3793, 3795. 3797, 3799, 3801, 3803, 3805,
3807, 3809, 3811, 3813, 3815, 3817, 3819, 3821, 3823, 3825, 3827,
3829, 3831, 3833, 3835, 3837, 3839, 3841, 3843, 3845, 3847, 3849,
3851, 3853, 3855, 3857, 3859, 3861, 3863, 3865 3887 and 3889 or a
fragment thereof.
26.-38. (canceled)
39. A method of evaluating Crohn's disease or detecting the
susceptibility to Crohn's disease comprising detecting at least one
mutation or polymorphism in Candidate Region 341 in a patient.
40.-48. (canceled)
49. The method of claim 39, wherein the mutation is at least one of
SNP from Candidate Region 341.
51. (canceled)
52. A method of evaluating Crohn's disease or diagnosing the
susceptibility to Crohn's disease in an individual, comprising
screening for an at-risk haplotype in Candidate Region 341, that is
more frequently present in an individual susceptible to Crohn's
disease compared to a control individual, wherein the presence of
the at-risk haplotype is indicative of an increased risk for the
onset or the development of Crohn's disease in the individual.
53. (canceled)
54. The method of claim 52, wherein the risk for the onset or the
development of Crohn's disease is increased at least about 20%.
55. The method of claim 52, wherein the at-risk haplotype is
characterized by the presence of at least one single nucleotide
polymorphism in Candidate Region 341.
56.-63. (canceled)
64. A method of determining a susceptibility to Crohn's disease in
an individual, comprising (a) determining the expression level or
the composition of a polypeptide encoded by at least one gene of
Candidate Region 341 in a test sample, (b) comparing the result
obtained in step (a) with the expression level or the composition
of the polypeptide encoded by the at least one gene in a control
sample to determine the presence of an alteration in the expression
level or the composition of the polypeptide in the test sample,
wherein the presence of the alteration in the expression level or
the composition of the polypeptide in the test sample is indicative
of an increased susceptibility to Crohn's Disease.
65. The method of claim 64, wherein the alteration in the
expression level or composition of the polypeptide is caused by the
expression of a splicing variant of a mRNA encoding the polypeptide
in the test sample that differs from a splicing variant expressed
in a control sample.
66. A drug screening assay comprising: (a) contacting a test
compound with a cell from an individual having Crohn's disease and
(b) determining the level of gene expression of at least one gene
in Candidate Region 341 in the cell of step (a) and (c) comparing
the level of gene expression obtained in step (b) with the level of
gene expression of the at least one gene in a normal cell; wherein
a similarity between the level of gene expression in the cell of
step (a) and the level of gene expression in the normal cell is
indicative that the test compound is a candidate drug to treat
Crohn's disease.
67.-68. (canceled)
69. A method for predicting the efficacy of a drug for treating
Crohn's disease in a human patient, comprising: (a) obtaining a a
gene expression profile from at least one of a gene in Candidate
Region 341 from a sample from the human patient to which the drug
has been administered and (b) comparing the gene expression profile
obtained in step (a) with a reference gene expression profile from
the at least one gene in Candidate Region 341, wherein a similarity
between the expression profile obtained in step (a) and the
reference expression profile is indicative of the efficacy of the
drug for treating Crohn's disease in the human patient.
70.-77. (canceled)
78. The method of claim 69, wherein the reference expression
profile is that of a cell derived from a patient that does not have
Crohn's disease.
79.-80. (canceled)
81. A method for predicting the efficacy of a drug for treating
Crohn's disease in a human patient, comprising: (a) obtaining a set
of genotypes from Candidate Region 341 from a sample of the human
patient to which the drug has been administered, and (b) comparing
the set of genotypes obtained in step (a) with a set of genotypes
from Candidate Region 341 associated with the efficacy of the drug,
wherein a similarity between the set of genotypes obtained in step
(a) and the set of genotypes associated with the efficacy of the
drug is indicative of the efficacy of the drug for treating Crohn's
disease in the human patient.
82.-85. (canceled)
86. The method of claim 81 wherein the set of genotypes of step (a)
is obtained by hybridization of allele-specific oligonucleotides
complementary to a polymorphic loci from any one of Tables 2, 3, 4,
5-36.
87. The method of claim 86, wherein the allele-specific
oligonucleotides comprise nucleic acid molecules at least 95%
identical to SEQ ID of any one of Tables 2, 3, 4, 5-36.
88.-117. (canceled)
118. A method for identifying a gene that regulates drug response
in Crohn's disease, comprising: (a) obtaining a gene expression
profile from at least one gene of Candidate Region 341 in a cell
induced to a pro-inflammatory like state in the presence of a
candidate drug; (b) obtaining a reference gene expression profile
for the at least one gene of Candidate region 341 in a cell induced
to a pro-inflammatory like state in the absence of the candidate
drug; and (c) comparing the gene expression profile obtained in
step (a) with the reference expression profile, wherein a
difference in the gene expression profiles is indicative that the
at least one gene regulates the candidate drug response Crohn's
disease.
119.-136. (canceled)
137. A method of assessing a patient's risk of having or developing
Crohn's disease, comprising: (a) in the patient's sample,
determining a level of expression of at least one gene or product
thereof of Candidate Region 341, (b) comparing the level of
expression obtained in step (a) to the level of expression of the
at least one gene or product thereof of a cell of an individual
suffering from Crohn's disease; and wherein a similarity between
the level of expression of step (a) and (b) is indicative of an
increased risk for the patient to have or develop Crohn's
disease.
138. A method of assessing a patient's risk of having or developing
Crohn's disease, comprising: (a) in the patient's sample,
determining a genotype for at least one polymorphic locus of
Candidate Region 341, (b) comparing said genotype obtained in step
(a) to a genotype for the at least one polymorphic locus of
Candidate Region 341 associated with Crohn's disease; and wherein a
similarity between the genotype of step (a) and (b) is indicative
of an increased risk for the patient to have or develop Crohn's
disease.
139.-140. (canceled)
141. A method of detecting the onset or the progression of Crohn's
disease in an individual, said method comprising comparing a
parameter from a sample from the individual to a control parameter,
wherein a difference between the parameter from the sample from the
individual and the control parameter is indicative of the onset or
progression of Crohn's disease in the individual and wherein the
parameter from the sample from the individual is selected from the
group consisting of: a level of expression of a gene in Candidate
Region 341; a mutation in a gene in Candidate Region 341; a single
nucleotide polymorphism in Candidate Region 341; a haplotype in
Candidate Region 341; a level of expression of a polypeptide
encoded by a gene in Candidate Region 341; and a mutation in a
polypeptide encoded by a gene in Candidate Region 341; and wherein
the control parameter is from a healthy individual and is selected
from the group consisting of: a level of expression of a gene in
Candidate Region 341; a mutation in a gene in Candidate Region 341;
a single nucleotide polymorphism in Candidate Region 341; a
haplotype in Candidate Region 341; a level of expression of a
polypeptide encoded by a gene in Candidate Region 341; and a
mutation in a polypeptide encoded by a gene in Candidate Region
341.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the field of genomics and genetics,
including genome analysis and the study of DNA variations. In
particular, the invention relates to the fields of
pharmacogenomics, diagnostics, patient therapy and the use of
genetic haplotype information to predict an individual's
susceptibility to inflammatory bowel disease (IBD), e.g. Crohn's
disease and/or their response to a particular drug or drugs, so
that drugs tailored to genetic differences of population groups may
be developed and/or administered to the appropriate population.
[0002] The invention also relates to a GeneMap for IBD (e.g.
Crohn's disease), which links variations in DNA (including both
genic and non-genic regions) to an individual's susceptibility to
IBD and/or response to a particular drug or drugs. The invention
further relates to the genes disclosed in the GeneMap (see Tables
2-4), which is related to methods and reagents for detection of an
individual's increased or decreased risk for Crohn's disease by
identifying at least one polymorphism in one or a combination of
the genes from the GeneMap. Also related are the candidate regions
identified in Table 1, which are associated with IBD. In addition,
the invention further relates to nucleotide sequences of those
genes including genomic DNA sequences, cDNA sequences, single
nucleotide polymorphisms (SNPs), other types of polymorphisms
(insertions, deletions, microsatellites), alleles and haplotypes
(see Sequence Listing and Tables 5-36).
[0003] The invention further relates to isolated nucleic acids
comprising these nucleotide sequences and isolated polypeptides or
peptides encoded thereby. Also related are expression vectors and
host cells comprising the disclosed nucleic acids or fragments
thereof, as well as antibodies that bind to the encoded
polypeptides or peptides.
[0004] The present invention further relates to ligands that
modulate the activity of the disclosed genes or gene products. In
addition, the invention relates to diagnostics and therapeutics for
IBD (in particular UC and Crohn's disease), utilizing the disclosed
nucleic acids, polymorphisms, chromosomal regions, gene maps,
polypeptides or peptides, antibodies and/or ligands and small
molecules that activate or repress relevant signaling events.
BACKGROUND OF THE INVENTION
[0005] Inflammatory bowel disease (IBD) is a collective term used
to describe two intestinal disorders whose etiology is not
completely understood: ex: Crohn's disease (CD) and ulcerative
colitis (UC). The course and prognosis of IBD, which occurs
worldwide and afflicts several million people, varies widely. Onset
of IBD is predominant in young adulthood. Symptoms of IBD include
abdominal cramps and pain, diarrhea, weight loss and intestinal
bleeding. Anemia and weight loss are also common signs of IBD.
Between 10% and 15% of people with IBD require surgery over a
ten-year period. Patients with IBD are also at increased risk for
the development of intestinal cancer. These diseases are
accompanied by a high frequency of psychological symptoms,
including anxiety and depression.
[0006] There are common features in many of the later stages of
IBD. Inflammation at the disease site/target organ is typically
present, caused by the release of inflammatory (also termed
"proinflammatory") cytokines by T cells and by other cells that
contribute to the activation steps and effector pathways of
immune/inflammatory processes. The current consensus opinion
regarding the pathogenesis of IBD centers on the role of
genetically determined dysregulation in the host immune response
toward the resident bacterial flora.
[0007] UC involves the rectum and spreads proximally to contiguous
portions or to the entire colon. Disease activity is usually
intermittent, with relapses and periods of quiescence. The
sigmoidoscopic or colonoscopic picture is characteristic. In mild
disease, the colonic mucosa appears hyperemic and granular. In more
severe disease, tiny punctuate ulcers are present and the mucosa is
characteristically friable and may bleed spontaneously.
Histologically, the inflammatory cell infiltrate in active disease
usually includes neutrophils, often invading crypts as well as
being associated with epithelial damage and crypt distortion. An
increased number of lymphocytes in the lamina propria and basal
plasmacytosis are usually present. Between 500,000 and 700,000
patients suffer from UC in the United States. Extra-colonic
manifestations of UC include arthritis, uveitis, aphthous
stomatitis, pyoderma gangrenosum, and erythema nodosum. Initial
therapy for patients with mild to moderate disease is usually an
aminosalicylate. In controlled trials, disease improvement by
various criteria occurred in up to 30% of subjects in the placebo
groups; thus, no specific treatment may be an option for patients
with very mild disease. In patients with active UC who do not
respond to standard 5-ASA treatment and in those with more severe
disease, oral corticosteroids have been the mainstay of acute
symptomatic therapy. However, corticosteroids are not effective in
long-term maintenance of remission in patients with UC given that
their use is associated with significant toxicity over time.
Although the pathogenesis of UC is not fully understood, there is
increasing evidence that UC may be an autoimmune disorder, with B
cells playing a role in disease pathophysiology. B cells, as well
as T cells, are present in basal lymphoid aggregates, a
histopathologic feature considered indicative of UC and seen in
histologic sections from patients with active UC. Whereas mucosal
inflammation in UC is thought to be driven by activated T cells,
these patients have a T-helper-2 (Th2) cytokine expression pattern
profile. As. Th2 cytokines classically drive B-cell immune
responses and antibody production, a central role for B cell may be
postulated in UC.
[0008] Crohn's disease is an Inflammatory Bowel Disease (IBD) in
which inflammation extends beyond the inner gut lining and
penetrates deeper layers of the intestinal wall of any part of the
digestive system (esophagus, stomach, small intestine, large
intestine, and/or anus). Crohn's disease is a chronic, lifelong
disease which can cause painful, often life altering symptoms
including diarrhea, cramping and rectal bleeding. Crohn's disease
occurs most frequently in the industrialized world and the typical
age of onset falls into two distinct ranges, 15 to 30 years of age
and 60 to 80 years of age. The highest mortality is during the
first years of disease, and in cases where the disease symptoms are
long lasting, an increased risk of colon cancer is observed.
Crohn's disease presently accounts for approximately two thirds of
IBD-related physician visits and hospitalizations, and 50 to 80% of
Crohn's disease patients eventually require surgical treatment.
Development of Crohn's disease is influenced by environmental and
host specific factors, together with "exogenous biological factors"
such as constituents of the intestinal flora (the naturally
occurring bacteria found in the intestine). It is believed that in
genetically predisposed individuals, exogenous factors such as
infectious agents, and host-specific characteristics such as
intestinal barrier function and/or blood supply, combine with
specific environmental factors to cause a chronic state of
improperly regulated immune system function. In this hypothetical
model, microorganisms trigger an immune response in the intestine,
and in susceptible individuals, this immune response is not turned
off when the microorganism is cleared from the body. The
chronically "turned on" immune response causes damage to the
intestine resulting in the symptoms of Crohn's disease.
[0009] Current treatments for Crohn's disease are primarily aimed
at reducing symptoms by suppressing inflammation and do not address
the root cause of the disease. Despite a preponderance of evidence
showing inheritance of a risk for Crohn's disease through
epidemiological studies and genome wide linkage analyses, the genes
affecting Crohn's disease have yet to be discovered (Hugot J P, and
Thomas G., 1998). There is a need in the art for identifying
specific genes related to Crohn's disease to enable the development
of therapeutics that address the causes of the disease rather than
relieving its symptoms. The failure in past studies to identify
causative genes in complex diseases, such as Crohn's disease, has
been due to the lack of appropriate methods to detect a sufficient
number of variations in genomic DNA samples (markers), the
insufficient quantity of necessary markers available, and the
number of needed individuals to enable such a study.
[0010] Unfortunately, new therapies for IBD are few, and both
diagnosis and treatment have been hampered by a lack of detailed
knowledge of the etiology. Despite the progress noted above, there
remains a need in the art for new and improved methods for treating
this debilitating group of diseases, and the present inventors have
made a significant step forward with the invention disclosed
herein. The invention includes a method for treating IBD and
inflammatory diseases in a subject, comprising administering to a
subject in need of such treatment an effective amount of a
pharmaceutical composition that comprises (a) an compound that
inhibits inflammation; and (b) a pharmaceutically acceptable
carrier, thereby treating the disease.
[0011] The DNA sequences between two human genomes are 99.9%
identical. The variations in DNA sequence between individuals can
be, as an example, deletions of small or large stretches of DNA,
insertions of stretches of DNA, variations in the number of
repetitive DNA elements, and changes in single base positions in
the genome called "single nucleotide polymorphisms" (SNPs). Human
DNA sequence variation accounts for a large fraction of observed
differences between individuals, including susceptibility to
disease.
[0012] Many common diseases, like IBD, are complex genetic traits
and are believed to involve several disease-genes rather than
single genes, as is observed for rare diseases. This makes
detection of any particular gene substantially more difficult than
in a rare disease, where a single gene mutation that segregates
according to a Mendelian inheritance pattern is the causative
mutation. Any one of the multiple interacting gene mutations
involved in the etiology of a complex disease will impart a lower
relative risk for the disease than will the single gene mutation
involved in a simple genetic disease. Low relative risk alleles are
more difficult to detect and, as a result, the success of
positional cloning using linkage mapping that was achieved for
simple genetic disease genes has not been repeated for complex
diseases.
[0013] Several approaches have been proposed to discover and
characterize multiple genes in complex genetic traits. These gene
discovery methods can be subdivided into hypothesis-free disorder
association studies and hypothesis-driven candidate gene or region
studies. The candidate gene approach relies on the analysis of a
gene in patients who have a disorder in which the gene is thought
to play a role. This approach is limited in utility because it only
provides for the investigation of genes with known functions.
Although variant sequences of candidate genes may be identified
using this approach, it is inherently limited by the fact that
variant sequences in other genes that contribute to the phenotype
will be necessarily missed when the technique is employed.
Genome-wide scans (GWS) have been shown to be efficient in
identifying disease genes, such as Crohn's disease susceptibility
genes (NOD2/CARD15 and OCTN). In contrast to the candidate gene
approach, a GWS searches throughout the genome without any a priori
hypothesis and consequently can identify genes that are not obvious
candidates for the disease as well as genes that are relevant
candidates for the disease it can also identify chromosomal regions
that are structurally important where mutations can influence gene
function of specific genes.
[0014] Family-based linkage mapping methods were initially used for
disorder locus identification. This technique locates genes based
on the relatively limited number of genetic recombination events
within the families used in the study, and results in large
chromosomal regions containing hundreds of genes, any one of which
could be the disorder-causing gene. Population-based, or linkage
disequilibrium (LD) mapping is based on the premise that regions
adjacent to a gene of interest are co-transmitted through the
generations along with the gene. As a result, LD extends over
shorter genetic regions than does linkage (Hewett et al., 2002),
and can facilitate detection of genes with lower relative risk than
family linkage mapping approaches. LD-based mapping also defines
much smaller candidate regions, which may contain only a few genes,
making the identification of the actual disorder gene much
easier.
[0015] It has been estimated that a GWS that uses a general
population and case/control association (LD) analysis would require
approximately 700,000 SNP markers (Carlson et al., 2003). The cost
of a GWS at this marker density for a sufficient sample size for
statistical power is economically prohibitive. The use of a special
founder population (genetic isolate), such as the French Canadian
population of Quebec, is one solution to the problem with LD
analysis. The French Canadian population in Quebec (Quebec Founder
Population--QFP) provides one of the best resources in the world
for gene discovery based on its high levels of genetic sharing and
genetic homogeneity. By combining DNA collected from the QFP, high
throughput genotyping capabilities and proprietary algorithms for
genetic analysis, a comprehensive genome-wide association study was
facilitated. The present invention relates specifically to a set of
IBD (E.g., Crohn's disease)-causing genes (GeneMap) and targets,
which present attractive points of therapeutic intervention.
[0016] In view of the foregoing, identifying susceptibility genes
associated with IBD (e.g. Crohn's disease) and their respective
biochemical pathways will facilitate the identification of
diagnostic markers as well as novel targets for improved
therapeutics. It will also improve the quality of life for those
afflicted by this disease and will reduce the economic costs of
these afflictions at the individual and societal level. The
identification of those genetic markers would provide the basis for
novel genetic tests and eliminate or reduce the therapeutic methods
currently used. The identification of those genetic markers will
also provide the development of effective therapeutic intervention
for the battery of laboratory, radiological, and endoscopic
evaluations typically required to diagnose IBD. The present
invention satisfies this need and provides related advantages as
well.
DESCRIPTION OF THE FILES CONTAINED ON THE CD-R
[0017] The contents of the submission on compact discs submitted
herewith are incorporated herein by reference in their entirety: A
compact disc copy of the Sequence Listing (COPY 1) (filename: GENI
011 01WO SeqList.txt, date recorded: Dec. 19, 2006, file size
39,614,000 bytes); a duplicate compact disc copy of the Sequence
Listing (COPY 2) (filename: GENI 011 01WO SeqList.txt, date
recorded: Dec. 19, 2006, file size 39,614,000 bytes); a duplicate
compact disc copy of the Sequence Listing (COPY 3) (filename: GENI
011 01WO SeqList.txt, date recorded: Dec. 19, 2006, file size
39,614,000 bytes); a computer readable format copy of the Sequence
Listing (CRF COPY) (filename: GENI 011 01WO SeqList.txt, date
recorded: Dec. 19, 2006, file size 39,614,000 bytes).
DEFINITIONS
[0018] Throughout the description of the present invention, several
terms are used that are specific to the science of this field. For
the sake of clarity and to avoid any misunderstanding, these
definitions are provided to aid in the understanding of the
specification and claims:
[0019] Allele: One of a pair, or series, of forms of a gene or
non-genic region that occur at a given locus in a chromosome.
Alleles are symbolized with the same basic symbol (e.g., B for
dominant and b for recessive; B1, B2, Bn for n additive alleles at
a locus). In a normal diploid cell there are two alleles of any one
gene (one from each parent), which occupy the same relative
position (locus) on homologous chromosomes. Within a population
there may be more than two alleles of a gene. See multiple alleles.
SNPs also have alleles, i.e., the two (or more) nucleotides that
characterize the SNP.
[0020] Amplification of nucleic acids: refers to methods such as
polymerase chain reaction (PCR), ligation amplification (or ligase
chain reaction, LCR) and amplification methods based on the use of
Q-beta replicase. These methods are well known in the art and are
described, for example, in U.S. Pat. Nos. 4,683,195 and 4,683,202.
Reagents and hardware for conducting PCR are commercially
available. Primers useful for amplifying sequences from the
disorder region are preferably complementary to, and preferably
hybridize specifically to, sequences in the disorder region or in
regions that flank a target region therein. Genes from Tables 2-4
generated by amplification may be sequenced directly.
Alternatively, the amplified sequence(s) may be cloned prior to
sequence analysis.
[0021] Antigenic component: is a moiety that binds to its specific
antibody with sufficiently high affinity to form a detectable
antigen-antibody complex.
[0022] Antibodies: refer to polyclonal and/or monoclonal antibodies
and fragments thereof, and immunologic binding equivalents thereof,
that can bind to proteins and fragments thereof or to nucleic acid
sequences from the disorder region, particularly from the disorder
gene products or a portion thereof. The term antibody is used both
to refer to a homogeneous molecular entity, or a mixture such as a
serum product made up of a plurality of different molecular
entities. Proteins may be prepared synthetically in a protein
synthesizer and coupled to a carrier molecule and injected over
several months into rabbits. Rabbit sera are tested for
immunoreactivity to the protein or fragment. Monoclonal antibodies
may be made by injecting mice with the proteins, or fragments
thereof. Monoclonal antibodies can be screened by ELISA and tested
for specific immunoreactivity with protein or fragments thereof
(Harlow et al. 1988, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.). These antibodies will
be useful in developing assays as well as therapeutics.
[0023] Associated allele: refers to an allele at a polymorphic
locus that is associated with a particular phenotype of interest,
e.g., a predisposition to a disorder or a particular drug
response.
[0024] cDNA: refers to complementary or copy DNA produced from an
RNA template by the action of RNA-dependent DNA polymerase (reverse
transcriptase). Thus, a cDNA clone means a duplex DNA sequence
complementary to an RNA molecule of interest, included in a cloning
vector or PCR amplified. This term includes genes from which the
intervening sequences have been removed.
[0025] cDNA library: refers to a collection of recombinant DNA
molecules containing cDNA inserts that together comprise
essentially all of the expressed genes of an organism or tissue. A
cDNA library can be prepared by methods known to one skilled in the
art (see, e.g., Cowell and Austin, 1997, "DNA Library Protocols,"
Methods in Molecular Biology). Generally, RNA is first isolated
from the cells of the desired organism, and the RNA is used to
prepare cDNA molecules.
[0026] Cloning: refers to the use of recombinant DNA techniques to
insert a particular gene or other DNA sequence into a vector
molecule. In order to successfully clone a desired gene, it is
necessary to use methods for generating DNA fragments, for joining
the fragments to vector molecules, for introducing the composite
DNA molecule into a host cell in which it can replicate, and for
selecting the clone having the target gene from amongst the
recipient host cells.
[0027] Cloning vector: refers to a plasmid or phage DNA or other
DNA molecule that is able to replicate in a host cell. The cloning
vector is typically characterized by one or more endonuclease
recognition sites at which such DNA sequences may be cleaved in a
determinable fashion without loss of an essential biological
function of the DNA, and which may contain a selectable marker
suitable for use in the identification of cells containing the
vector.
[0028] Coding sequence or a protein-coding sequence: is a
polynucleotide sequence capable of being transcribed into mRNA
and/or capable of being translated into a polypeptide or peptide.
The boundaries of the coding sequence are typically determined by a
translation start codon at the 5'-terminus and a translation stop
codon at the 3'-terminus.
[0029] Complement of a nucleic acid sequence: refers to the
antisense sequence that participates in Watson-Crick base-pairing
with the original sequence.
[0030] Disorder region: refers to the portions of the human
chromosomes displayed in Table 1 bounded by the markers from Tables
2-36.
[0031] Disorder-associated nucleic acid or polypeptide sequence:
refers to a nucleic acid sequence that maps to region of Table 1 or
the polypeptides encoded therein (Tables 2-4, nucleic acids, and
polypeptides). For nucleic acids, this encompasses sequences that
are identical or complementary to the gene sequences from Tables
2-4, as well as sequence-conservative, function-conservative, and
non-conservative variants thereof. For polypeptides, this
encompasses sequences that are identical to the polypeptide, as
well as function-conservative and non-conservative variants
thereof. Included are the alleles of naturally-occurring
polymorphisms causative of IBD such as, but not limited to, alleles
that cause altered expression of genes of Tables 2-4 and alleles
that cause altered protein levels or stability (e.g., decreased
levels, increased levels, expression in an inappropriate tissue
type, increased stability, and decreased stability).
[0032] Expression vector: refers to a vehicle or plasmid that is
capable of expressing a gene that has been cloned into it, after
transformation or integration in a host cell. The cloned gene is
usually placed under the control of (i.e., operably linked to) a
regulatory sequence.
[0033] Function-conservative variants: are those in which a change
in one or more nucleotides in a given codon position results in a
polypeptide sequence in which a given amino acid residue in the
polypeptide has been replaced by a conservative amino acid
substitution. Function-conservative variants also include analogs
of a given polypeptide and any polypeptides that have the ability
to elicit antibodies specific to a designated polypeptide.
[0034] Founder population: Also called a population isolate, this
is a large number of people who have mostly descended, in genetic
isolation from other populations, from a much smaller number of
people who lived many generations ago.
[0035] Gene: Refers to a DNA sequence that encodes through its
template or messenger RNA a sequence of amino acids characteristic
of a specific peptide, polypeptide, or protein. The term "gene"
also refers to a DNA sequence that encodes an RNA product. The term
gene as used herein with reference to genomic DNA includes
intervening, non-coding regions, as well as regulatory regions, and
can include 5' and 3' ends. A gene sequence is wild-type if such
sequence is usually found in individuals unaffected by the disorder
or condition of interest. However, environmental factors and other
genes can also play an important role in the ultimate determination
of the disorder. In the context of complex disorders involving
multiple genes (oligogenic disorder), the wild type, or normal
sequence can also be associated with a measurable risk or
susceptibility, receiving its reference status based on its
frequency in the general population.
[0036] GeneMaps: are defined as groups of gene(s) that are directly
or indirectly involved in at least one phenotype of a disorder
(some non-limiting example of GeneMaps comprises various
combinations of genes from Tables 2-4). As such, GeneMaps enable
the development of synergistic diagnostic products, creating
"theranostics".
[0037] Genotype: Set of alleles at a specified locus or loci.
[0038] Haplotype: The allelic pattern of a group of (usually
contiguous) DNA markers or other polymorphic loci along an
individual chromosome or double helical DNA segment. Haplotypes
identify individual chromosomes or chromosome segments. The
presence of shared haplotype patterns among a group of individuals
implies that the locus defined by the haplotype has been inherited,
identical by descent, from a common ancestor. Detection of
identical by descent haplotypes is the basis of linkage
disequilibrium (LD) mapping. Haplotypes are broken down through the
generations by recombination and mutation. In some instances, a
specific allele or haplotype may be associated with susceptibility
to a disorder or condition of interest, e.g., Crohn's disease. In
other instances, an allele or haplotype may be associated with a
decrease in susceptibility to a disorder or condition of interest,
i.e., a protective sequence.
[0039] Host: includes prokaryotes and eukaryotes. The term includes
an organism or cell that is the recipient of an expression vector
(e.g., autonomously replicating or integrating vector).
[0040] Hybridizable: nucleic acids are hybridizable to each other
when at least one strand of the nucleic acid can anneal to another
nucleic acid strand under defined stringency conditions. In some
embodiments, hybridization requires that the two nucleic acids
contain at least 10 substantially complementary nucleotides;
depending on the stringency of hybridization, however, mismatches
may be tolerated. The appropriate stringency for hybridizing
nucleic acids depends on the length of the nucleic acids and the
degree of complementarity, and can be determined in accordance with
the methods described herein.
[0041] Identity by descent: Identity among DNA sequences for
different individuals that is due to the fact that they have all
been inherited from a common ancestor. LD mapping identifies
Identity by descent haplotypes as the likely location of disorder
genes shared by a group of patients.
[0042] Identity: as known in the art, is a relationship between two
or more polypeptide sequences or two or more polynucleotide
sequences, as determined by comparing the sequences. In the art,
identity also means the degree of sequence relatedness between
polypeptide or polynucleotide sequences, as the case may be, as
determined by the match between strings of such sequences. Identity
and similarity can be readily calculated by known methods,
including but not limited to those described in A. M. Lesk (ed),
1988, Computational Molecular Biology, Oxford University Press, NY;
D. W. Smith (ed), 1993, Biocomputing. Informatics and Genome
Projects, Academic Press, NY; A. M. Griffin and H. G. Griffin, H. G
(eds), 1994, Computer Analysis of Sequence Data, Part 1, Humana
Press, NJ; G. von Heinje, 1987, Sequence Analysis in Molecular
Biology, Academic Press; and M. Gribskov and J. Devereux (eds),
1991, Sequence Analysis Primer, M Stockton Press, NY; H. Carillo
and D. Lipman, 1988, SIAM J. Applied Math., 48:1073.
[0043] Immunogenic component: is a moiety that is capable of
eliciting a humoral and/or cellular immune response in a host
animal.
[0044] Isolated nucleic acids: are nucleic acids separated away
from other components (e.g., DNA, RNA, and protein) with which they
are associated (e.g., as obtained from cells, chemical synthesis
systems, or phage or nucleic acid libraries). Isolated nucleic
acids are at least 60% free, preferably 75% free, and most
preferably 90% free from other associated components. In accordance
with the present invention, isolated nucleic acids can be obtained
by methods described herein, or other established methods,
including isolation from natural sources (e.g., cells, tissues, or
organs), chemical synthesis, recombinant methods, combinations of
recombinant and chemical methods, and library screening
methods.
[0045] Isolated polypeptides or peptides: are those that are
separated from other components (e.g., DNA, RNA, and other
polypeptides or peptides) with which they are associated (e.g., as
obtained from cells, translation systems, or chemical synthesis
systems). In a preferred embodiment, isolated polypeptides or
peptides are at least 10% pure; more preferably, 80% or 90% pure.
Isolated polypeptides and peptides include those obtained by
methods described herein, or other established methods, including
isolation from natural sources (e.g., cells, tissues, or organs),
chemical synthesis, recombinant methods, or combinations of
recombinant and chemical methods. Proteins or polypeptides referred
to herein as recombinant are proteins or polypeptides produced by
the expression of recombinant nucleic acids. A portion as used
herein with regard to a protein or polypeptide, refers to fragments
of that protein or polypeptide. The fragments can range in size
from 5 amino acid residues to all but one residue of the entire
protein sequence. Thus, a portion or fragment can be at least 5,
5-50, 50-100, 100-200, 200-400, 400-800, or more consecutive amino
acid residues of a protein or polypeptide.
[0046] Linkage disequilibrium (LD): the situation in which the
alleles for two or more loci do not occur together in individuals
sampled from a population at frequencies predicted by the product
of their individual allele frequencies. In other words, markers
that are in LD do not follow Mendel's second law of independent
random segregation. LD can be caused by any of several demographic
or population artifacts as well as by the presence of genetic
linkage between markers. However, when these artifacts are
controlled and eliminated as sources of LD, then LD results
directly from the fact that the loci involved are located close to
each other on the same chromosome so that specific combinations of
alleles for different markers (haplotypes) are inherited together.
Markers that are in high LD can be assumed to be located near each
other and a marker or haplotype that is in high LD with a genetic
trait can be assumed to be located near the gene that affects that
trait. The physical proximity of markers can be measured in family
studies where it is called linkage or in population studies where
it is called linkage disequilibrium.
[0047] LD mapping: population based gene mapping, which locates
disorder genes by identifying regions of the genome where
haplotypes or marker variation patterns are shared statistically
more frequently among disorder patients compared to healthy
controls. This method is based upon the assumption that many of the
patients will have inherited an allele associated with the disorder
from a common ancestor (IBD), and that this allele will be in LD
with the disorder gene.
[0048] Locus: a specific position along a chromosome or DNA
sequence. Depending upon context, a locus could be a gene, a
marker, a chromosomal band or a specific sequence of one or more
nucleotides.
[0049] Minor allele frequency (MAF): the population frequency of
one of the alleles for a given polymorphism, which is equal or less
than 50%. The sum of the MAF and the Major allele frequency equals
one.
[0050] Markers: an identifiable DNA sequence that is variable
(polymorphic) for different individuals within a population. These
sequences facilitate the study of inheritance of a trait or a gene.
Such markers are used in mapping the order of genes along
chromosomes and in following the inheritance of particular genes;
genes closely linked to the marker or in LD with the marker will
generally be inherited with it. Two types of markers are commonly
used in genetic analysis, microsatellites and SNPs.
[0051] Microsatellite: DNA of eukaryotic cells comprising a
repetitive, short sequence of DNA that is present as tandem repeats
and in highly variable copy number, flanked by sequences unique to
that locus.
[0052] Mutant sequence: if it differs from one or more wild-type
sequences. For example, a nucleic acid from a gene listed in Tables
2-4 containing a particular allele of a single nucleotide
polymorphism may be a mutant sequence. In some cases, the
individual carrying this allele has increased susceptibility toward
the disorder or condition of interest. In other cases, the mutant
sequence might also refer to an allele that decreases the
susceptibility toward a disorder or condition of interest and thus
acts in a protective manner. The term mutation may also be used to
describe a specific allele of a polymorphic locus.
[0053] Non-conservative variants: are those in which a change in
one or more nucleotides in a given codon position results in a
polypeptide sequence in which a given amino acid residue in a
polypeptide has been replaced by a non-conservative amino acid
substitution. Non-conservative variants also include polypeptides
comprising non-conservative amino acid substitutions.
[0054] Nucleic acid or polynucleotide: purine- and
pyrimidine-containing polymers of any length, either
polyribonucleotides or polydeoxyribonucleotide or mixed polyribo
polydeoxyribonucleotides. This includes single- and double-stranded
molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as
protein nucleic acids (PNA) formed by conjugating bases to an amino
acid backbone. This also includes nucleic acids containing modified
bases.
[0055] Nucleotide: a nucleotide, the unit of a DNA molecule, is
composed of a base, a 2'-deoxyribose and phosphate ester(s)
attached at the 5' carbon of the deoxyribose. For its incorporation
in DNA, the nucleotide needs to possess three phosphate esters but
it is converted into a monoester in the process.
[0056] Operably linked: means that the promoter controls the
initiation of expression of the gene. A promoter is operably linked
to a sequence of proximal DNA if upon introduction into a host cell
the promoter determines the transcription of the proximal DNA
sequence(s) into one or more species of RNA. A promoter is operably
linked to a DNA sequence if the promoter is capable of initiating
transcription of that DNA sequence.
[0057] Ortholog: denotes a gene or polypeptide obtained from one
species that has homology to an analogous gene or polypeptide from
a different species.
[0058] Paralog: denotes a gene or polypeptide obtained from a given
species that has homology to a distinct gene or polypeptide from
that same species.
[0059] Phenotype: any visible, detectable or otherwise measurable
property of an organism such as symptoms of, or susceptibility to,
a disorder.
[0060] Polymorphism: occurrence of two or more alternative genomic
sequences or alleles between or among different genomes or
individuals at a single locus. A polymorphic site thus refers
specifically to the locus at which the variation occurs. In some
cases, an individual carrying a particular allele of a polymorphism
has an increased or decreased susceptibility toward a disorder or
condition of interest.
[0061] Portion and fragment: are synonymous. A portion as used with
regard to a nucleic acid or polynucleotide refers to fragments of
that nucleic acid or polynucleotide. The fragments can range in
size from 8 nucleotides to all but one nucleotide of the entire
gene sequence. Preferably, the fragments are at least about 8 to
about 10 nucleotides in length; at least about 12 nucleotides in
length; at least about 15 to about 20 nucleotides in length; at
least about 25 nucleotides in length; or at least about 35 to about
55 nucleotides in length.
[0062] Probe or primer: refers to a nucleic acid or oligonucleotide
that forms a hybrid structure with a sequence in a target region of
a nucleic acid due to complementarity of the probe or primer
sequence to at least one portion of the target region sequence.
[0063] Protein and polypeptide: are synonymous. Peptides are
defined as fragments or portions of polypeptides, preferably
fragments or portions having at least one functional activity
(e.g., proteolysis, adhesion, fusion, antigenic, or intracellular
activity) as the complete polypeptide sequence.
[0064] Recombinant nucleic acids: nucleic acids which have been
produced by recombinant DNA methodology, including those nucleic
acids that are generated by procedures which rely upon a method of
artificial replication, such as the polymerase chain reaction (PCR)
and/or cloning into a vector using restriction enzymes. Portions of
recombinant nucleic acids which code for polypeptides can be
identified and isolated by, for example, the method of M. Jasin et
al., U.S. Pat. No. 4,952,501.
[0065] Regulatory sequence: refers to a nucleic acid sequence that
controls or regulates expression of structural genes when operably
linked to those genes. These include, for example, the lac systems,
the trp system, major operator and promoter regions of the phage
lambda, the control region of fd coat protein and other sequences
known to control the expression of genes in prokaryotic or
eukaryotic cells. Regulatory sequences will vary depending on
whether the vector is designed to express the operably linked gene
in a prokaryotic or eukaryotic host, and may contain
transcriptional elements such as enhancer elements, termination
sequences, tissue-specificity elements and/or translational
initiation and termination sites.
[0066] Sample: as used herein refers to a biological sample, such
as, for example, tissue or fluid isolated from an individual or
animal (including, without limitation, plasma, serum, cerebrospinal
fluid, lymph, tears, nails, hair, saliva, milk, pus, and tissue
exudates and secretions) or from in vitro cell
culture-constituents, as well as samples obtained from, for
example, a laboratory procedure.
[0067] Single nucleotide polymorphism (SNP): variation of a single
nucleotide. This includes the replacement of one nucleotide by
another and deletion or insertion of a single nucleotide.
Typically, SNPs are biallelic markers although tri- and
tetra-allelic markers also exist. For example, SNP A\C may comprise
allele C or allele A (Tables 5-36). Thus, a nucleic acid molecule
comprising SNP A\C may include a C or A at the polymorphic
position. For clarity purposes, an ambiguity code is used in Tables
5-36 and the sequence listing, to represent the variations. For a
combination of SNPs, the term "haplotype" is used, e.g. the
genotype of the SNPs in a single DNA strand that are linked to one
another. In certain embodiments, the term "haplotype" is used to
describe a combination of SNP alleles, e.g., the alleles of the
SNPs found together on a single DNA molecule. In specific
embodiments, the SNPs in a haplotype are in linkage disequilibrium
with one another.
[0068] Sequence-conservative: variants are those in which a change
of one or more nucleotides in a given codon position results in no
alteration in the amino acid encoded at that position (i.e., silent
mutation).
[0069] Substantially homologous: a nucleic acid or fragment thereof
is substantially homologous to another if, when optimally aligned
(with appropriate nucleotide insertions and/or deletions) with the
other nucleic acid (or its complementary strand), there is
nucleotide sequence identity in at least 60% of the nucleotide
bases, usually at least 70%, more usually at least 80%, preferably
at least 90%, and more preferably at least 95-98% of the nucleotide
bases. Alternatively, substantial homology exists when a nucleic
acid or fragment thereof will hybridize, under selective
hybridization conditions, to another nucleic acid (or a
complementary strand thereof). Selectivity of hybridization exists
when hybridization which is substantially more selective than total
lack of specificity occurs. Typically, selective hybridization will
occur when there is at least about 55% sequence identity over a
stretch of at least about nine or more nucleotides, preferably at
least about 65%, more preferably at least about 75%, and most
preferably at least about 90% (M. Kanehisa, 1984, NucL Acids Res.
11:203-213). The length of homology comparison, as described, may
be over longer stretches, and in certain embodiments will often be
over a stretch of at least 14 nucleotides, usually at least 20
nucleotides, more usually at least 24 nucleotides, typically at
least 28 nucleotides, more typically at least 32 nucleotides, and
preferably at least 36 or more nucleotides.
[0070] Wild-type gene from Tables 2-4: refers to the reference
sequence. The wild-type gene sequences from Tables 2-36 used to
identify the variants (polymorphisms, alleles, and haplotypes)
described in detail herein.
[0071] Technical and scientific terms used herein have the meanings
commonly understood by one of ordinary skill in the art to which
the present invention pertains, unless otherwise defined. Reference
is made herein to various methodologies known to those of skill in
the art. Publications and other materials setting forth such known
methodologies to which reference is made are incorporated herein by
reference in their entireties as though set forth in full. Standard
reference works setting forth the general principles of recombinant
DNA technology include J. Sambrook et al., 1989, Molecular Cloning:
A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.; P. B. Kaufman et al., (eds), 1995,
Handbook of Molecular and Cellular Methods in Biology and Medicine,
CRC Press, Boca Raton; M. J. McPherson (ed), 1991, Directed
Mutagenesis: A Practical Approach, IRL Press, Oxford; J. Jones,
1992, Amino Acid and Peptide Synthesis, Oxford Science
Publications, Oxford; B. M. Austen and O. M. R. Westwood, 1991,
Protein Targeting and Secretion, IRL Press, Oxford; D. N Glover
(ed), 1985, DNA Cloning, Volumes I and 11; M. J. Gait (ed), 1984,
Oligonucleotide Synthesis; B. D. Hames and S. J. Higgins (eds),
1984, Nucleic Acid Hybridization; Quirke and Taylor (eds), 1991,
PCR-A Practical Approach; Harries and Higgins (eds), 1984,
Transcription and Translation; R. I. Freshney (ed), 1986, Animal
Cell Culture; Immobilized Cells and Enzymes, 1986, IRL Press;
Perbal, 1984, A Practical Guide to Molecular Cloning, J. H. Miller
and M. P. Calos (eds), 1987, Gene Transfer Vectors for Mammalian
Cells, Cold Spring Harbor Laboratory Press; M. J. Bishop (ed),
1998, Guide to Human Genome Computing, 2d Ed., Academic Press, San
Diego, Calif.; L. F. Peruski and A. H. Peruski, 1997, The Internet
and the New Biology. Tools for Genomic and Molecular Research,
American Society for Microbiology, Washington, D.C. Standard
reference works setting forth the general principles of immunology
include S. Sell, 1996, Immunology, Immunopathology & Immunity,
5th Ed., Appleton & Lange, Publ., Stamford, Conn.; D. Male et
al., 1996, Advanced Immunology, 3d Ed., Times Mirror Int'l
Publishers Ltd., Publ., London; D. P. Stites and A. L Terr, 1991,
Basic and Clinical Immunology, 7th Ed., Appleton & Lange,
Publ., Norwalk, Conn.; and A. K. Abbas et al., 1991, Cellular and
Molecular Immunology, W. B. Saunders Co., Publ., Philadelphia, Pa.
Any suitable materials and/or methods known to those of skill can
be utilized in carrying out the present invention; however,
preferred materials and/or methods are described. Materials,
reagents, and the like to which reference is made in the following
description and examples are generally obtainable from commercial
sources, and specific vendors are cited herein.
DETAILED DESCRIPTION OF THE INVENTION
[0072] Inflammatory Bowel Disease (IBD) is characterized by
excessive and chronic inflammation at various sites in the
gastrointestinal tract. IBD describes two clinical conditions
called Crohn's disease (CD) and ulcerative colitis (UC). CD and UC
share many clinical and pathological characteristics but they also
have some markedly different features. There is strong scientific
support suggesting that the main pathological processes in these
two diseases are distinct. This patent application will focus
primarily on IBD (ex: UC and CD), but is not limited to IBD and
encompasses inflammatory diseases in general for therapeutic
targets, since IBD and inflammatory diseases can share common
therapeutic targets.
Previously Identified Genes and Loci
[0073] Genetic studies have previously indicated the presence of
several loci predisposing to Inflammatory Bowel Diseases. Nine IBD
loci have been identified: IBD1 (CARD15/NOD2, Caspase recruitment
domain family, member 15 (NOD2 protein)), IBD2 (Inflammatory bowel
disease-2), IBD3 (Inflammatory bowel disease-3), IBD4 (Inflammatory
bowel disease-4), IBD5 (Inflammatory bowel disease-5), IBD6
(Inflammatory bowel disease-6), IBD7 (Inflammatory bowel
disease-7), IBD8 (Inflammatory bowel disease-8), and IBD9
(Inflammatory bowel disease 9).
[0074] Several loci have been identified and replicated to date;
they are located on chromosomes 16q12, 12q13.2-q24.1, 6p21,
14q11-q12, 5q31-q33, 19p13, 1p36, 16p, and 3p26 respectively (Wild
and Rioux 2004; Duerr et al., 2002). Results from linkage studies
have suggested that CD and UC share some loci but do not share
others, such as locus 16q12 which is unique to CD (The IBD
International Genetics Consortium 2001). Most of the genes
determining susceptibility in each of these chromosomal regions
remain to be identified. The most widely replicated loci are on
chromosomes 16 (caused by mutations in the NOD2/CARD15 gene), 12,
6, 14 and 5 (OCTN2 genes at IBD5 locus), which predispose to
early-onset Crohn's disease. Thus, there is a continuing need in
the medical arts for genetic markers of IBD and guidance for the
use of such markers.
[0075] Several years ago, two different groups reported the
association of CARD15 (NOD2) variants with CD (Hugot 2001; Ogura
2001). CARD15 is located at the IBD1 locus. The gene codes for an
intracellular receptor involved in the innate immune detection of
bacterial products. This detection induces the activation of the
NF-kB pathway which is of particular importance in immune and
inflammatory responses (Philpott and Viala 2004). Three major
mutations represent 82% of the total CARD15 mutations: R702W,
G908R, and 1007fsinsC. However they do not explain over 20% of the
genetic predisposition to the disease, and altogether, they are
carried by 30-50% of CD patients and 15-20% of healthy controls in
Caucasian populations. These values are much lower in Japanese or
Africans (reviewed in Girardin 2003).
[0076] Recently, the OCTN1 and OCTN2 genes at IBD5 locus have been
associated with CD (Peltekova 2004). Variants in these genes are in
strong linkage disequilibrium and create a two-allele risk
haplotype enriched in patients with CD. Both proteins are
trans-membrane sodium-dependent carnitine transporters and
sodium-independent organic cation transporters. The variants may
cause disease by impairing OCTN activity or expression, reducing
carnitine transport in a cell-type and disease-specific manner
(Peltekova 2004). The IBD5 locus contains multiple candidate genes
including the genes for organic cation transporters (OCTN2/SLC22A4
and SLC22A5), the gene for a LIN4-domain-containing protein
(RILIPDLIM3), the gene for the oc2 subunit of proline hydroxylase
(P4HA2) and a gene of unknown function (NCBI UniGene identifier
Hs.70932). Because of extensive linkage disequilibrium (LD) in this
region, it has not been possible to further refine the SNP map and
unambiguously identify a single susceptibility gene. With respect
to OCTN2, the basal transcription is downregulated by the C allele
of G-207C, and this allele disrupts the ability of SLC22A5 to be
upregulated in response to heat shock or arachidonic acid as a
result of impaired HSF I binding and subsequent transcriptional
activation.
[0077] The mechanism by which these organic cation transporters
contribute to the pathology of Crohn's disease may relate to the in
vivo metabolic importance of carnitine. Carnitine facilitates
transport of long chain fatty acids across the mitochondrial inner
membrane for subsequent P-oxidation, and is also important in the
maintenance of cellular CoenzymeA levels. Carnitine uptake into
lymphocytes, along with a corresponding decrease in plasma levels,
is a physiological response to inflammation. Symptoms of the
related condition Ulcerative Colitis may be due to an energy
deficiency in colonic epithelium secondary to poor mitochondrial
function due to decreased long-chain fatty acid transport to the
mitochondria (Roediger W E 1980). The haplotype of mutations in
Crohn's disease patients provided previously might affect cellular
metabolic energy levels in inflamed tissue by combining impaired
OCTN1 transporter function with downregulation and inability to
respond to heat or inflammatory stress by the OCTN2 gene. Heat
shock proteins and arachidonic acids are involved in response to a
variety of cellular stresses, including sepsis, metabolic stress
and ischaemia. The heat shock response modulates inflammation
through modulation of NF-kB activation. OCTN2 is a heat stress
inducible protein. Thus, the OCTN2 gene is normally upregulated in
response to inflammation through binding of HSF I protein to its
promoter. This in turn mobilizes carnitine and bolsters metabolism
in the inflamed tissue. Impaired OCTN1 carnitine transporter
activity and lowered OCTN2 expression level results in reduced
metabolism and either triggers or worsens cellular stress in areas
of inflammation. The two OCTN transporters may therefore function
in the inflammatory pathology of Crohn's disease. Additionally,
OCTN1 is a polyspecific cation transporter, and might have a role
in the uptake of drugs used to treat CD from the gut. As described
above, OCTN2 is a transporter protein with the ability to transport
carnitine in a sodium dependent manner. Missense mutations and
nonsense mutations in the organic cation transporter OCTN2 had
previously been identified in patients with primary Systemic
Carnitine Deficiency (SCD), an autosomal recessive disorder
characterized by progressive cardiomyopathy, skeletal myopathy,
hypoglycemia and hyperammonemia.
[0078] Genes encoding proteins involved in the immune system,
epithelial functions, and host response to micro-organisms
represent good potential candidates for IBD and have been examined
in numerous case-control studies. However, many of the published
associations of genetic variants with IBD have not been replicated
in follow-up studies.
[0079] The genetic variants that have been identified so far in IBD
explain only a fraction of the genetic predisposition to this
disorder. It is clear that multiple components contribute to
disease risk, each component having a modest effect on disease
susceptibility. Thus the development of GeneMaps for IBD may lead
to a better understanding of pathogenesis and to the identification
of new pathways involved in the disease, ultimately leading to
better treatments for the patients. GeneMaps may also lead to
molecular diagnostic tools that will identify subjects at risk for
CD or for serious complications of the disease.
Genome Wide Association Study to Construct a GeneMap for IBD (Ex:
Crohn's Disease)
[0080] The present invention is based on the discovery of genes
associated with IBD and inflammatory diseases in general. In the
preferred embodiment, disease-associated loci (candidate regions;
Table 1) are identified by the statistically significant
differences in allele or haplotype frequencies between the cases
and the controls. For the purpose of the present invention, 654
candidate regions (Table 1) are identified.
[0081] The invention provides a method for the discovery of genes
associated with IBD (E.g. Crohn's disease) and the construction of
a GeneMap for Crohn's disease in a human population, comprising the
following steps (see Example section herein):
Step 1: Recruit Patients (Cases) and Controls
[0082] In the preferred embodiment, 500 patients diagnosed for
Crohn's disease along with two family members are recruited from
the Quebec Founder Population (QFP). The preferred trios recruited
are parent-parent-child (PPC) trios. Trios can also be recruited as
parent-child-child (PCC) trios. In another preferred embodiment,
more or less than 500 trios are recruited. In another embodiment,
independent case and control samples are recruited.
[0083] In another embodiment, the present invention is performed as
a whole or partially with DNA samples from individuals of another
founder population than the Quebec population or from the general
population.
Step 2: DNA Extraction and Quantitation
[0084] Any sample comprising cells or nucleic acids from patients
or controls may be used. Preferred samples are those easily
obtained from the patient or control. Such samples include, but are
not limited to blood, peripheral lymphocytes, buccal swabs,
epithelial cell swabs, nails, hair, bronchoalveolar lavage fluid,
sputum, or other body fluid or tissue obtained from an
individual.
[0085] In one embodiment, DNA is extracted from such samples in the
quantity and quality necessary to perform the invention using
conventional DNA extraction and quantitation techniques. The
present invention is not linked to any DNA extraction or
quantitation platform in particular.
Step 3: Genotype the Recruited Individuals
[0086] In one embodiment, assay-specific and/or locus-specific
and/or allele-specific oligonucleotides for every SNP marker of the
present invention (Tables 5-36) are organized onto one or more
arrays. The genotype at each SNP locus is revealed by hybridizing
short PCR fragments comprising each SNP locus onto these arrays.
The arrays permit a high-throughput genome wide association study
using DNA samples from individuals of the Quebec founder
population. Such assay-specific and/or locus-specific and/or
allele-specific oligonucleotides necessary for scoring each SNP of
the present invention are preferably organized onto a solid
support. Such supports can be arrayed on wafers, glass slides,
beads or any other type of solid support.
[0087] In another embodiment, the assay-specific and/or
locus-specific and/or allele-specific oligonucleotides are not
organized onto a solid support but are still used as a whole, in
panels or one by one. The present invention is therefore not linked
to any genotyping platform in particular.
[0088] In another embodiment, one or more portions of the SNP maps
(publicly available maps, proprietary maps from Perlegen Sciences,
Inc. (Mountain View, Calif., USA), and our own proprietary QLDM
map) are used to screen the whole genome, a subset of chromosomes,
a chromosome, a subset of genomic regions or a single genomic
region.
[0089] The 1,500 individuals composing the 500 trios are preferably
individually genotyped with at least 80,000 markers, generating at
least a few million genotypes; more preferably, at least a hundred
million.
Step 4: Exclude the Markers that Did not Pass the Quality Control
of the Assay.
[0090] Preferably, the quality controls consist of, but are not
limited to, the following criteria: eliminate SNPs that had a high
rate of Mendelian errors (cut-off at 1% Mendelian error rate), that
deviate from the Hardy-Weinberg equilibrium, that are
non-polymorphic in the Quebec founder population or have too many
missing data (cut-off at 1% missing values or higher), or simply
because they are non-polymorphic in the Quebec founder population
(cut-off at 1%<10% minor allele frequency (MAF)).
Step 5: Perform the Genetic Analysis on the Results Obtained Using
Haplotype Information as Well as Single-Marker Association.
[0091] In the preferred embodiment, genetic analysis is performed
on all the genotypes from step 3.
[0092] In another embodiment, genetic analysis is performed on a
total of 248,535 SNPs.
[0093] In one embodiment, the genetic analysis consists of, but is
not limited to features corresponding to Phase information and
haplotype structures. Phase information and haplotype structures
are preferably deduced from trio genotypes using Phasefinder. Since
chromosomal assignment (phase) cannot be estimated when all trio
members are heterozygous, an Expectation-Maximization (EM)
algorithm may be used to resolve chromosomal assignment ambiguities
after Phasefinder.
[0094] In yet another embodiment, the PL-EM algorithm
(Partition-Ligation E M; Niu et al., Am. J. Hum. Genet. 70:157
(2002)) can be used to estimate haplotypes from the "genotype" data
as a measured estimate of the reference allele frequency of a SNP
in 15-marker windows that advance in increments of one marker
across the data set. The results from such algorithms are converted
into 15-marker haplotype files. Subsequently, the individual
15-marker block files are assembled into one continuous block of
haplotypes for the entire chromosome. These extended haplotypes can
then be used for further analysis. Such haplotype assembly
algorithms take the consensus estimate of the allele call at each
marker over all separate estimations (most markers are estimated 15
different times as the 15 marker blocks pass over their
position).
[0095] In the preferred embodiment, the haplotypes for both the
controls and the patients are derived in this manner. The preferred
control of a trio structure is the spouse if the patient is one of
the parents or the non-transmitted chromosomes (chromosomes found
in parents but not in affected child) if the patient is the
child.
[0096] In another embodiment, the haplotype frequencies among
patients are compared to those among the controls using LDSTATS, a
program that assesses the association of haplotypes with the
disease. Such program defines haplotypes using multi-marker windows
that advance across the marker map in one-marker increments. Such
windows can be 1, 3, 5, 7 or 9 markers wide, and all these window
sizes are tested concurrently. Larger multi-marker haplotype
windows can also be used. At each position the frequency of
haplotypes in cases is compared to the frequency of haplotypes in
controls. Such allele frequency differences for single marker
windows can be tested using Pearson's Chi-square with any degree of
freedom. Multi-allelic haplotype association can be tested using
Smith's normalization of the square root of Pearson's Chi-square.
Such significance of association can be reported in two ways:
[0097] The significance of association within any one haplotype
window is plotted against the marker that is central to that
window.
[0098] P-values of association for each specific marker are
calculated as a pooled P-value across all haplotype windows in
which they occur. The pooled P-value is calculated using an
expected value and variance calculated using a permutation test
that considers covariance between individual windows. Such pooled
P-values can yield narrower regions of gene location than the
window data (see Example 3 for details on analysis methods, such as
LDSTATS v2.0 and v4.0).
[0099] In another embodiment, conditional haplotype analyses can be
performed on subsets of the original set of cases and controls
using the program LDSTATS. The selection of a subset of cases and
their matched controls can be based on the carrier status of cases
at a gene or locus of interest. Various conditional haplotypes can
be derived, such as protective haplotypes and risk haplotypes.
Step 6: Fine Mapping
[0100] In this step, the candidate regions that were identified by
step 4 are further mapped for the purpose of refinement and
validation.
[0101] In the preferred embodiment, this fine mapping is performed
with a density of genetic markers higher than in the genome wide
scan (step 3) using any genotyping platform available in the art.
Such fine mapping can be, but is not limited to, typing the allele
via an allele-specific elongation assay that is then ligated to a
locus-specific oligonucleotide. Such assays can be performed
directly on the genomic DNA at a highly multiplex level and the
products can be amplified using universal oligonucleotides. For
each candidate region, the density of genetic markers can be, but
is not limited to, a set of SNP markers with an average
inter-marker distance of 1-4 Kb distributed over about 400 Kb to 1
Mb, roughly centered at the highest point of the GWS association.
The preferred samples are those obtained from Crohn's disease PPC
trios including the ones used for the GWS. Other preferred samples
are trios or case control samples from another population, such as
a General population.
[0102] In the preferred embodiment, the genetic analysis of the
results obtained using haplotype information as well as
single-marker association (as performed as in step 5, described
herein) is performed as described herein (step 5 and Example
section). The candidate regions that are validated and confirmed
after this analysis proceed to a gene mining step described in
Example 5, herein, to characterize their marker and genetic
content.
Step 7: SNP and DNA Polymorphism Discovery
[0103] In the preferred embodiment, all the candidate genes and
regions identified in step 6 are sequenced for polymorphism
identification.
[0104] In another embodiment, the entire region, including all
introns, is sequenced to identify all polymorphisms.
[0105] In yet another embodiment, the candidate genes are
prioritized for sequencing, and only functional gene elements
(promoters, conserved noncoding sequences, exons and splice sites)
are sequenced.
[0106] In yet another embodiment, previously identified
polymorphisms in the candidate regions can also be used. For
example, SNPs from dbSNP, Perlegen Sciences, Inc., or others can
also be used rather than resequencing the candidate regions to
identify polymorphisms.
[0107] The discovery of SNPs and DNA polymorphisms generally
comprises a step consisting of determining the major haplotypes in
the region to be sequenced. The preferred samples are selected
according to which haplotypes contribute to the association signal
observed in the region to be sequenced. The purpose is to select a
set of samples that covers all the major haplotypes in the given
region. Each major haplotype is preferably analyzed in at least a
few individuals.
[0108] Any analytical procedure may be used to detect the presence
or absence of variant nucleotides at one or more polymorphic
positions of the invention. In general, the detection of allelic
variation requires a mutation discrimination technique, optionally
an amplification reaction and optionally a signal generation
system. Any means of mutation detection or discrimination may be
used. For instance, DNA sequencing, scanning methods,
hybridization, extension based methods, incorporation based
methods, restriction enzyme-based methods and ligation-based
methods may be used in the methods of the invention.
[0109] Sequencing methods include, but are not limited to, direct
sequencing, and sequencing by hybridization. Scanning methods
include, but are not limited to, protein truncation test (PTT),
single-strand conformation polymorphism analysis (SSCP), denaturing
gradient gel electrophoresis (DGGE), temperature gradient gel
electrophoresis (TGGE), cleavage, heteroduplex analysis, chemical
mismatch cleavage (CMC), and enzymatic mismatch cleavage.
Hybridization-based methods of detection include, but are not
limited to, solid phase hybridization such as dot blots, multiple
allele specific diagnostic assay (MASDA), reverse dot blots, and
oligonucleotide arrays (DNA Chips). Solution phase hybridization
amplification methods may also be used, such as Taqman. Extension
based methods include, but are not limited to, amplification
refraction mutation systems (ARMS), amplification refractory
mutation systems (ALEX), and competitive oligonucleotide priming
systems (COPS). Incorporation based methods include, but are not
limited to, mini-sequencing and arrayed primer extension (APEX).
Restriction enzyme-based detection systems include, but are not
limited to, restriction site generating PCR. Lastly, ligation based
detection methods include, but are not limited to, oligonucleotide
ligation assays (OLA). Signal generation or detection systems that
may be used in the methods of the invention include, but are not
limited to, fluorescence methods such as fluorescence resonance
energy transfer (FRET), fluorescence quenching, fluorescence
polarization as well as other chemiluminescence,
electrochemiluminescence, Raman, radioactivity, colometric methods,
hybridization protection assays and mass spectrometry methods.
Further amplification methods include, but are not limited to self
sustained replication (SSR), nucleic acid sequence based
amplification (NASBA), ligase chain reaction (LCR), strand
displacement amplification (SDA) and branched DNA (B-DNA).
Step 8: Ultrafine Mapping
[0110] This step further maps the candidate regions and genes
confirmed in the previous step to identify and validate the
responsible polymorphisms associated with Crohn's disease in the
human population.
[0111] In a preferred embodiment, the discovered SNPs and
polymorphisms of step 7 are ultrafine mapped at a higher density of
markers than the fine mapping described herein using the same
technology described in step 6.
Step 9: GeneMap Construction
[0112] The confirmed variations in DNA (including both genic and
non-genic regions) are used to build a GeneMap for IBD (ex: Crohn's
disease). The gene content of this GeneMap is described in more
detail below. Such GeneMap can be used for other methods of the
invention comprising the diagnostic methods described herein, the
susceptibility to Crohn's disease, the response to a particular
drug, the efficacy of a particular drug, the screening methods
described herein and the treatment methods described herein.
[0113] As is evident to one of ordinary skill in the art, all of
the above steps or the steps do not need to be performed, or
performed in a given order to practice or use the SNPs, genomic
regions, genes, proteins, etc. in the methods of the invention.
Genes from the GeneMap
[0114] In one embodiment the GeneMap consists of genes and targets,
in a variety of combinations, identified from the candidate regions
listed in Table 1. In another embodiment, all genes from Tables 2-4
are present in the GeneMap. In another preferred embodiment, the
GeneMap consists of a selection of genes from Tables 2-4. The genes
of the invention (Tables 2-4) are arranged by candidate regions and
by their chromosomal location. Such order is for the purpose of
clarity and does not reflect any other criteria of selection in the
association of the genes with IBD (ex: Crohn's disease).
[0115] In the preferred embodiment, genes identified in the WGAS
and subsequent fine mapping studies for IBD (ex: Crohn's disease)
are evaluated using the Ingenuity Pathway Analysis application
(IPA, Ingenuity systems) in order to identify direct biological
interactions between these genes, and also to identify molecular
regulators acting on those genes (indirect interactions) that could
be also involved in IBD (ex: Crohn's disease). The purpose of this
effort is to decipher the molecules involved in contributing to IBD
(ex: Crohn's disease). These gene interaction networks are very
valuable tools in the sense that they facilitate extension of the
map of gene products that could represent potential drug targets
for IBD (ex: Crohn's disease).
Nucleic Acid Sequences
[0116] The nucleic acid sequences of the present invention may be
derived from a variety of sources including DNA, cDNA, synthetic
DNA, synthetic RNA, derivatives, mimetics or combinations thereof.
Such sequences may comprise genomic DNA, which may or may not
include naturally occurring introns, genic regions, nongenic
regions, and regulatory regions. Moreover, such genomic DNA may be
obtained in association with promoter regions or poly (A)
sequences. The sequences, genomic DNA, or cDNA may be obtained in
any of several ways. Genomic DNA can be extracted and purified from
suitable cells by means well known in the art. Alternatively, mRNA
can be isolated from a cell and used to produce cDNA by reverse
transcription or other means. The nucleic acids described herein
are used in certain embodiments of the methods of the present
invention for production of RNA, proteins or polypeptides, through
incorporation into cells, tissues, or organisms. In one embodiment,
DNA containing all or part of the coding sequence for the genes
described in Tables 2-4, or the SNP markers described in Tables
5-36, is incorporated into a vector for expression of the encoded
polypeptide in suitable host cells. The invention also comprises
the use of the nucleotide sequence of the nucleic acids of this
invention to identify DNA probes for the genes described in Tables
2-4 or the SNP markers described in Tables 5-36, PCR primers to
amplify the genes described in Tables 2-4 or the SNP markers
described in Tables 5-36, nucleotide polymorphisms in the genes
described in Tables 2-4, and regulatory elements of the genes
described in Tables 2-4. The nucleic acids of the present invention
find use as primers and templates for the recombinant production of
IBD-associated peptides or polypeptides, for chromosome and gene
mapping, to provide antisense sequences, for tissue distribution
studies, to locate and obtain full length genes, to identify and
obtain homologous sequences (wild-type and mutants), and in
diagnostic applications.
Antisense Oligonucleotides
[0117] In a particular embodiment of the invention, an antisense
nucleic acid or oligonucleotide is wholly or partially
complementary to, and can hybridize with, a target nucleic acid
(either DNA or RNA) having the sequence of SEQ ID NO:1, NO:3 or any
SEQ ID from any Tables of the invention. For example, an antisense
nucleic acid or oligonucleotide comprising 16 nucleotides can be
sufficient to inhibit expression of at least one gene from Tables
2-4. Alternatively, an antisense nucleic acid or oligonucleotide
can be complementary to 5' or 3' untranslated regions, or can
overlap the translation initiation codon (5' untranslated and
translated regions) of at least one gene from Tables 2-4, or its
functional equivalent. In another embodiment, the antisense nucleic
acid is wholly or partially complementary to, and can hybridize
with, a target nucleic acid that encodes a polypeptide from a gene
described in Tables 2-4.
[0118] In addition, oligonucleotides can be constructed which will
bind to duplex nucleic acid (i.e., DNA:DNA or DNA:RNA), to form a
stable triple helix containing or triplex nucleic acid. Such
triplex oligonucleotides can inhibit transcription and/or
expression of a gene from Tables 2-4, or its functional equivalent
(M. D. Frank-Kamenetskii et al., 1995). Triplex oligonucleotides
are constructed using the basepairing rules of triple helix
formation and the nucleotide sequence of the genes described in
Tables 2-4.
[0119] The present invention encompasses methods of using
oligonucleotides in antisense inhibition of the function of the
genes from Tables 2-4. In the context of this invention, the term
"oligonucleotide" refers to naturally-occurring species or
synthetic species formed from naturally-occurring subunits or their
close homologs. The term may also refer to moieties that function
similarly to oligonucleotides, but have non-naturally-occurring
portions. Thus, oligonucleotides may have altered sugar moieties or
inter-sugar linkages.
[0120] Exemplary among these are phosphorothioate and other sulfur
containing species, which are known in the art. In preferred
embodiments, at least one of the phosphodiester bonds of the
oligonucleotide has been substituted with a structure that
functions to enhance the ability of the compositions to penetrate
into the region of cells where the RNA whose activity is to be
modulated is located. It is preferred that such substitutions
comprise phosphorothioate bonds, methyl phosphonate bonds, or short
chain alkyl or cycloalkyl structures. In accordance with other
preferred embodiments, the phosphodiester bonds are substituted
with structures which are, at once, substantially non-ionic and
non-chiral, or with structures which are chiral and
enantiomerically specific. Persons of ordinary skill in the art
will be able to select other linkages for use in the practice of
the invention. Oligonucleotides may also include species that
include at least some modified base forms. Thus, purines and
pyrimidines other than those normally found in nature may be so
employed. Similarly, modifications on the furanosyl portions of the
nucleotide subunits may also be effected, as long as the essential
tenets of this invention are adhered to. Examples of such
modifications are 2'-O-alkyl- and 2'-halogen-substituted
nucleotides. Some non-limiting examples of modifications at the 2'
position of sugar moieties which are useful in the present
invention include OH, SH, SCH3, F, OCH3, OCN, O(CH2), NH2 and
O(CH2)n CH3, where n is from 1 to about 10. Such oligonucleotides
are functionally interchangeable with natural oligonucleotides or
synthesized oligonucleotides, which have one or more differences
from the natural structure. All such analogs are comprehended by
this invention so long as they function effectively to hybridize
with at least one gene from Tables 2-4 DNA or RNA to inhibit the
function thereof.
[0121] The oligonucleotides in accordance with this invention
preferably comprise from about 3 to about 50 subunits. It is more
preferred that such oligonucleotides and analogs comprise from
about 8 to about 25 subunits and still more preferred to have from
about 12 to about 20 subunits. As defined herein, a "subunit" is a
base and sugar combination suitably bound to adjacent subunits
through phosphodiester or other bonds. Antisense nucleic acids or
oligonucleotides can be produced by standard techniques (see, e.g.,
Shewmaker et al., U.S. Pat. No. 6,107,065). The oligonucleotides
used in accordance with this invention may be conveniently and
routinely made through the well-known technique of solid phase
synthesis. Any other means for such synthesis may also be employed;
however, the actual synthesis of the oligonucleotides is well
within the abilities of the practitioner. It is also well known to
prepare other oligonucleotides such as phosphorothioates and
alkylated derivatives.
[0122] The oligonucleotides of this invention are designed to be
hybridizable with RNA (e.g., mRNA) or DNA from genes described in
Tables 2-4. For example, an oligonucleotide (e.g., DNA
oligonucleotide) that hybridizes to mRNA from a gene described in
Tables 2-4 can be used to target the mRNA for RnaseH digestion.
Alternatively an oligonucleotide that can hybridize to the
translation initiation site of the mRNA of a gene described in
Tables 2-4 can be used to prevent translation of the mRNA. In
another approach, oligonucleotides that bind to the double-stranded
DNA of a gene from Tables 2-4 can be administered. Such
oligonucleotides can form a triplex construct and inhibit the
transcription of the DNA encoding polypeptides of the genes
described in Tables 2-4. Triple helix pairing prevents the double
helix from opening sufficiently to allow the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using triplex DNA have been described (see,
e.g., J. E. Gee et al., 1994, Molecular and Immunologic Approaches,
Futura Publishing Co., Mt. Kisco, N.Y.).
[0123] As non-limiting examples, antisense oligonucleotides may be
targeted to hybridize to the following regions: mRNA cap region;
translation initiation site; translational termination site;
transcription initiation site; transcription termination site;
polyadenylation signal; 3' untranslated region; 5' untranslated
region; 5' coding region; mid coding region; and 3' coding region.
Preferably, the complementary oligonucleotide is designed to
hybridize to the most unique 5' sequence of a gene described in
Tables 2-4, including any of about 15-35 nucleotides spanning the
5' coding sequence. In accordance with the present invention, the
antisense oligonucleotide can be synthesized, formulated as a
pharmaceutical composition, and administered to a subject. The
synthesis and utilization of antisense and triplex oligonucleotides
have been previously described (e.g., Simon et al., 1999; Barre et
al., 2000; Elez et al., 2000; Sauter et al., 2000).
[0124] Alternatively, expression vectors derived from retroviruses,
adenovirus, herpes or vaccinia viruses or from various bacterial
plasmids may be used for delivery of nucleotide sequences to the
targeted organ, tissue or cell population. Methods which are well
known to those skilled in the art can be used to construct
recombinant vectors which will express nucleic acid sequence that
is complementary to the nucleic acid sequence encoding a
polypeptide from the genes described in Tables 2-4. These
techniques are described both in Sambrook et al., 1989 and in
Ausubel et al., 1992. For example, expression of at least one gene
from Tables 2-4 can be inhibited by transforming a cell or tissue
with an expression vector that expresses high levels of
untranslatable sense or antisense sequences. Even in the absence of
integration into the DNA, such vectors may continue to transcribe
RNA molecules until they are disabled by endogenous nucleases.
Transient expression may last for a month or more with a
nonreplicating vector, and even longer if appropriate replication
elements are included in the vector system. Various assays may be
used to test the ability of gene-specific antisense
oligonucleotides to inhibit the expression of at least one gene
from Tables 2-4. For example, mRNA levels of the genes described in
Tables 2-4 can be assessed by Northern blot analysis (Sambrook et
al., 1989; Ausubel et al., 1992; J. C. Alwine et al. 1977; I. M.
Bird, 1998), quantitative or semi-quantitative RT-PCR analysis
(see, e.g., W. M. Freeman et al., 1999; Ren et al., 1998; J. M.
Cale et al., 1998), or in situ hybridization (reviewed by A. K.
Raap, 1998). Alternatively, antisense oligonucleotides may be
assessed by measuring levels of the polypeptide from the genes
described in Tables 2-4, e.g., by western blot analysis, indirect
immunofluorescence and immunoprecipitation techniques (see, e.g.,
J. M. Walker, 1998, Protein Protocols on CD-ROM, Humana Press,
Totowa, N.J.). Any other means for such detection may also be
employed, and is well within the abilities of the practitioner.
Mapping Technologies
[0125] The present invention includes various methods, which employ
mapping technologies to map SNPs and polymorphisms. For purpose of
clarity, this section comprises, but is not limited to, the
description of mapping technologies that can be utilized to achieve
the embodiments described herein. Mapping technologies may be based
on amplification methods, restriction enzyme cleavage methods,
hybridization methods, sequencing methods, and cleavage methods
using agents.
[0126] Amplification methods include: self sustained sequence
replication (Guatelli et al., 1990), transcriptional amplification
system (Kwoh et al., 1989), Q-Beta Replicase (Lizardi et al.,
1988), isothermal amplification (e.g. Dean et al., 2002; and Hafner
et al., 2001), or any other nucleic acid amplification method,
followed by the detection of the amplified molecules using
techniques well known to those of ordinary skill in the art. These
detection schemes are especially useful for the detection of
nucleic acid molecules if such molecules are present in very low
number.
[0127] Restriction enzyme cleavage methods include: isolating
sample and control DNA, amplification (optional), digestion with
one or more restriction endonucleases, determination of fragment
length sizes by gel electrophoresis and comparing samples and
controls. Differences in fragment length sizes between sample and
control DNA indicates mutations in the sample DNA. Moreover,
sequence specific ribozymes (see, e.g., U.S. Pat. No. 5,498,531) or
DNAzyme (e.g. U.S. Pat. No. 5,807,718) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
or DNAzyme cleavage site.
[0128] Hybridization methods include any measurement of the
hybridization or gene expression levels, of sample nucleic acids to
probes corresponding to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 50, 75, 100, 200, 500, 1000 or more genes, or ranges of
these numbers, such as about 2-10, about 10-20, about 20-50, about
50-100, about 100-200, about 200-500 or about 500-1000 genes of
Tables 2-4.
[0129] SNPs and SNP maps of the invention can be identified or
generated by hybridizing sample nucleic acids, e.g., DNA or RNA, to
high density arrays or bead arrays containing oligonucleotide
probes corresponding to the polymorphisms of Tables 5-36 (see the
Affymetrix arrays and Illumina bead sets at www.affymetrix.com and
www.illumina.com and see Cronin et al., 1996; or Kozal et al.,
1996).
[0130] Methods of forming high density arrays of oligonucleotides
with a minimal number of synthetic steps are known. The
oligonucleotide analogue array can be synthesized on a single or on
multiple solid substrates by a variety of methods, including, but
not limited to, light-directed chemical coupling, and mechanically
directed coupling (see Pirrung, U.S. Pat. No. 5,143,854).
[0131] In brief, the light-directed combinatorial synthesis of
oligonucleotide arrays on a glass surface proceeds using automated
phosphoramidite chemistry and chip masking techniques. In one
specific implementation, a glass surface is derivatized with a
silane reagent containing a functional group, e.g., a hydroxyl or
amine group blocked by a photolabile protecting group. Photolysis
through a photolithographic mask is used selectively to expose
functional groups which are then ready to react with incoming 5'
photoprotected nucleoside phosphoramidites. The phosphoramidites
react only with those sites which are illuminated (and thus exposed
by removal of the photolabile blocking group). Thus, the
phosphoramidites only add to those areas selectively exposed from
the preceding step. These steps are repeated until the desired
array of sequences have been synthesized on the solid surface.
Combinatorial synthesis of different oligonucleotide analogues at
different locations on the array is determined by the pattern of
illumination during synthesis and the order of addition of coupling
reagents.
[0132] In addition to the foregoing, additional methods which can
be used to generate an array of oligonucleotides on a single
substrate are described in PCT Publication Nos. WO 93/09668 and WO
01/23614. High density nucleic acid arrays can also be fabricated
by depositing pre-made or natural nucleic acids in predetermined
positions. Synthesized or natural nucleic acids are deposited on
specific locations of a substrate by light directed targeting and
oligonucleotide directed targeting. Another embodiment uses a
dispenser that moves from region to region to deposit nucleic acids
in specific spots.
[0133] Nucleic acid hybridization simply involves contacting a
probe and target nucleic acid under conditions where the probe and
its complementary target can form stable hybrid duplexes through
complementary base pairing. See WO 99/32660. The nucleic acids that
do not form hybrid duplexes are then washed away leaving the
hybridized nucleic acids to be detected, typically through
detection of an attached detectable label. It is generally
recognized that nucleic acids are denatured by increasing the
temperature or decreasing the salt concentration of the buffer
containing the nucleic acids. Under low stringency conditions
(e.g., low temperature and/or high salt) hybrid duplexes (e.g.,
DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed
sequences are not perfectly complementary. Thus, specificity of
hybridization is reduced at lower stringency. Conversely, at higher
stringency (e.g., higher temperature or lower salt) successful
hybridization tolerates fewer mismatches. One of skill in the art
will appreciate that hybridization conditions may be selected to
provide any degree of stringency.
[0134] In a preferred embodiment, hybridization is performed at low
stringency, in this case in 6.times.SSPET at 37.degree. C. (0.005%
Triton X-100), to ensure hybridization and then subsequent washes
are performed at higher stringency (e.g., 1.times.SSPET at
37.degree. C.) to eliminate mismatched hybrid duplexes. Successive
washes may be performed at increasingly higher stringency (e.g.,
down to as low as 0.25.times.SSPET at 37.degree. C. to 50.degree.
C.) until a desired level of hybridization specificity is obtained.
Stringency can also be increased by addition of agents such as
formamide. Hybridization specificity may be evaluated by comparison
of hybridization to the test probes with hybridization to the
various controls that can be present (e.g., expression level
control, normalization control, mismatch controls, etc.).
[0135] In general, there is a tradeoff between hybridization
specificity (stringency) and signal intensity. Thus, in a preferred
embodiment, the wash is performed at the highest stringency that
produces consistent results and that provides a signal intensity
greater than approximately 10% of the background intensity. Thus,
in a preferred embodiment, the hybridized array may be washed at
successively higher stringency solutions and read between each
wash. Analysis of the data sets thus produced will reveal a wash
stringency above which the hybridization pattern is not appreciably
altered and which provides adequate signal for the particular
oligonucleotide probes of interest.
[0136] Probes based on the sequences of the genes described above
may be prepared by any commonly available method. Oligonucleotide
probes for screening or assaying a tissue or cell sample are
preferably of sufficient length to specifically hybridize only to
appropriate, complementary genes or transcripts. Typically the
oligonucleotide probes will be at least about 10, 12, 14, 16, 18,
20 or 25 nucleotides in length. In some cases, longer probes of at
least 30, 40, or 50 nucleotides will be desirable.
[0137] As used herein, oligonucleotide sequences that are
complementary to one or more of the genes or gene fragments
described in Tables 2-4 refer to oligonucleotides that are capable
of hybridizing under stringent conditions to at least part of the
nucleotide sequences of said genes. Such hybridizable
oligonucleotides will typically exhibit at least about 75% sequence
identity at the nucleotide level to said genes, preferably about
80% or 85% sequence identity or more preferably about 90% or 95% or
more sequence identity to said genes (see GeneChip.RTM. Expression
Analysis Manual, Affymetrix, Rev. 3, which is herein incorporated
by reference in its entirety).
[0138] The phrase "hybridizing specifically to" or "specifically
hybridizes" refers to the binding, duplexing, or hybridizing of a
molecule substantially to or only to a particular nucleotide
sequence or sequences under stringent conditions when that sequence
is present in a complex mixture (e.g., total cellular) DNA or
RNA.
[0139] As used herein a "probe" is defined as a nucleic acid,
capable of binding to a target nucleic acid of complementary
sequence through one or more types of chemical bonds, usually
through complementary base pairing, usually through hydrogen bond
formation. As used herein, a probe may include natural (i.e., A, G,
U, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In
addition, the bases in probes may be joined by a linkage other than
a phosphodiester bond, so long as it does not interfere with
hybridization. Thus, probes may be peptide nucleic acids in which
the constituent bases are joined by peptide bonds rather than
phosphodiester linkages.
[0140] A variety of sequencing reactions known in the art can be
used to directly sequence nucleic acids for the presence or the
absence of one or more polymorphisms of Tables 5-36. Examples of
sequencing reactions include those based on techniques developed by
Maxam and Gilbert (1977) or Sanger (1977). It is also contemplated
that any of a variety of automated sequencing procedures can be
utilized, including sequencing by mass spectrometry (see, e.g. PCT
International Publication No. WO 94/16101; Cohen et al., 1996; and
Griffin et al., 1993), real-time pyrophosphate sequencing method
(Ronaghi et al., 1998; and Permutt et al., 2001) and sequencing by
hybridization (see e.g. Drmanac et al., 2002).
[0141] Other methods of detecting polymorphisms include methods in
which protection from cleavage agents is used to detect mismatched
bases in RNA/RNA, DNA/DNA or RNA/DNA heteroduplexes (Myers et al.,
1985). In general, the technique of "mismatch cleavage" starts by
providing heteroduplexes formed by hybridizing (labeled) RNA or DNA
containing a wild-type sequence with potentially mutant RNA or DNA
obtained from a sample. The double-stranded duplexes are treated
with an agent who cleaves single-stranded regions of the duplex
such as which will exist due to basepair mismatches between the
control and sample strands. For instance, RNA/DNA duplexes can be
treated with RNase and DNA/DNA hybrids treated with S1 nuclease to
enzymatically digest the mismatched regions. In other embodiments,
either DNA/DNA or RNA/DNA duplexes can be treated with
hydroxylamine or osmium tetroxide and with piperidine in order to
digest mismatched regions. After digestion of the mismatched
regions, the resulting material is then separated by size on
denaturing polyacrylamide gels to determine the site of a mutation
or SNP (see, for example, Cotton et al., 1988; and Saleeba et al.,
1992). In a preferred embodiment, the control DNA or RNA can be
labeled for detection.
[0142] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping polymorphisms. For
example, the mutY enzyme of E. coli cleaves A at G/A mismatches
(Hsu et al., 1994). Other examples include, but are not limited to,
the MutHLS enzyme complex of E. coli (Smith and Modrich Proc. 1996)
and Cel 1 from the celery (Kulinski et al., 2000) both cleave the
DNA at various mismatches. According to an exemplary embodiment, a
probe based on a polymorphic site corresponding to a polymorphism
of Tables 5-6 is hybridized to a cDNA or other DNA product from a
test cell or cells. The duplex is treated with a DNA mismatch
repair enzyme, and the cleavage products, if any, can be detected
from electrophoresis protocols or the like. See, for example, U.S.
Pat. No. 5,459,039. Alternatively, the screen can be performed in
vivo following the insertion of the heteroduplexes in an
appropriate vector. The whole procedure is known to those ordinary
skilled in the art and is referred to as mismatch repair detection
(see e.g. Fakhrai-Rad et al., 2004).
[0143] In other embodiments, alterations in electrophoretic
mobility can be used to identify polymorphisms in a sample. For
example, single strand conformation polymorphism (SSCP) analysis
can be used to detect differences in electrophoretic mobility
between mutant and wild type nucleic acids (Orita et al., 1989;
Cotton et al., 1993; and Hayashi 1992). Single-stranded DNA
fragments of case and control nucleic acids will be denatured and
allowed to renature. The secondary structure of single-stranded
nucleic acids varies according to sequence. The resulting
alteration in electrophoretic mobility enables the detection of
even a single base change. The DNA fragments may be labeled or
detected with labeled probes. The sensitivity of the assay may be
enhanced by using RNA (rather than DNA), in which the secondary
structure is more sensitive to a change in sequence. In a preferred
embodiment, the method utilizes heteroduplex analysis to separate
double stranded heteroduplex molecules on the basis of changes in
electrophoretic mobility (Kee et al., 1991).
[0144] In yet another embodiment, the movement of mutant or
wild-type fragments in a polyacrylamide gel containing a gradient
of denaturant is assayed using denaturing gradient gel
electrophoresis (DGGE) (Myers et al., 1985). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum et al., 1987). In another
embodiment, the mutant fragment is detected using denaturing HPLC
(see e.g. Hoogendoorn et al., 2000).
[0145] Examples of other techniques for detecting polymorphisms
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, selective primer extension,
selective ligation, single-base extension, selective termination of
extension or invasive cleavage assay. For example, oligonucleotide
primers may be prepared in which the polymorphism is placed
centrally and then hybridized to target DNA under conditions which
permit hybridization only if a perfect match is found (Saiki et
al., 1986; Saiki et al., 1989). Such oligonucleotides are
hybridized to PCR amplified target DNA or a number of different
mutations when the oligonucleotides are attached to the hybridizing
membrane and hybridized with labeled target DNA. Alternatively, the
amplification, the allele-specific hybridization and the detection
can be done in a single assay following the principle of the 5'
nuclease assay (e.g. see Livak et al., 1995). For example, the
associated allele, a particular allele of a polymorphic locus, or
the like is amplified by PCR in the presence of both
allele-specific oligonucleotides, each specific for one or the
other allele. Each probe has a different fluorescent dye at the 5'
end and a quencher at the 3' end. During PCR, if one or the other
or both allele-specific oligonucleotides are hybridized to the
template, the Taq polymerase via its 5' exonuclease activity will
release the corresponding dyes. The latter will thus reveal the
genotype of the amplified product.
[0146] Hybridization assays may also be carried out with a
temperature gradient following the principle of dynamic
allele-specific hybridization or like e.g. Jobs et al., (2003); and
Bourgeois and Labuda, (2004). For example, the hybridization is
done using one of the two allele-specific oligonucleotides labeled
with a fluorescent dye, and an intercalating quencher under a
gradually increasing temperature. At low temperature, the probe is
hybridized to both the mismatched and full-matched template. The
probe melts at a lower temperature when hybridized to the template
with a mismatch. The release of the probe is captured by an
emission of the fluorescent dye, away from the quencher. The probe
melts at a higher temperature when hybridized to the template with
no mismatch. The temperature-dependent fluorescence signals
therefore indicate the absence or presence of an associated allele,
a particular allele of a polymorphic locus, or the like (e.g. Jobs
et al., 2003). Alternatively, the hybridization is done under a
gradually decreasing temperature. In this case, both
allele-specific oligonucleotides are hybridized to the template
competitively. At high temperature none of the two probes are
hybridized. Once the optimal temperature of the full-matched probe
is reached, it hybridizes and leaves no target for the mismatched
probe (e.g. Bourgeois and Labuda, 2004). In the latter case, if the
allele-specific probes are differently labeled, then they are
hybridized to a single PCR-amplified target. If the probes are
labeled with the same dye, then the probe cocktail is hybridized
twice to identical templates with only one labeled probe, different
in the two cocktails, in the presence of the unlabeled competitive
probe.
[0147] Alternatively, allele specific amplification technology that
depends on selective PCR amplification may be used in conjunction
with the present invention. Oligonucleotides used as primers for
specific amplification may carry the associated allele, a
particular allele of a polymorphic locus, or the like, also
referred to as "mutation" of interest in the center of the
molecule, so that amplification depends on differential
hybridization (Gibbs et al., 1989) or at the extreme 3' end of one
primer where, under appropriate conditions, mismatch can prevent,
or reduce polymerase extension (Prossner, 1993). In addition it may
be desirable to introduce a novel restriction site in the region of
the mutation to create cleavage-based detection (Gasparini et al.,
1992). It is anticipated that in certain embodiments, amplification
may also be performed using Taq ligase for amplification (Barany,
1991). In such cases, ligation will occur only if there is a
perfect match at the 3' end of the 5' sequence making it possible
to detect the presence of a known associated allele, a particular
allele of a polymorphic locus, or the like at a specific site by
looking for the presence or absence of amplification. The products
of such an oligonucleotide ligation assay can also be detected by
means of gel electrophoresis. Furthermore, the oligonucleotides may
contain universal tags used in PCR amplification and zip code tags
that are different for each allele. The zip code tags are used to
isolate a specific, labeled oligonucleotide that may contain a
mobility modifier (e.g. Grossman et al., 1994).
[0148] In yet another alternative, allele-specific elongation
followed by ligation will form a template for PCR amplification. In
such cases, elongation will occur only if there is a perfect match
at the 3' end of the allele-specific oligonucleotide using a DNA
polymerase. This reaction is performed directly on the genomic DNA
and the extension/ligation products are amplified by PCR. To this
end, the oligonucleotides contain universal tags allowing
amplification at a high multiplex level and a zip code for SNP
identification. The PCR tags are designed in such a way that the
two alleles of a SNP are amplified by different forward primers,
each having a different dye. The zip code tags are the same for
both alleles of a given SNPs and they are used for hybridization of
the PCR-amplified products to oligonucleotides bound to a solid
support, chip, bead array or like. For an example of the procedure,
see Fan et al. (Cold Spring Harbor Symposia on Quantitative
Biology, Vol. LXVIII, pp. 69-78 2003).
[0149] Another alternative includes the single-base
extension/ligation assay using a molecular inversion probe,
consisting of a single, long oligonucleotide (see e.g. Hardenbol et
al., 2003). In such an embodiment, the oligonucleotide hybridizes
on both side of the SNP locus directly on the genomic DNA, leaving
a one-base gap at the SNP locus. The gap-filling, one-base
extension/ligation is performed in four tubes, each having a
different dNTP. Following this reaction, the oligonucleotide is
circularized whereas unreactive, linear oligonucleotides are
degraded using an exonuclease such as exonuclease I of E. coli. The
circular oligonucleotides are then linearized and the products are
amplified and labeled using universal tags on the oligonucleotides.
The original oligonucleotide also contains a SNP-specific zip code
allowing hybridization to oligonucleotides bound to a solid
support, chip, and bead array or like. This reaction can be
performed at a high multiplexed level.
[0150] In another alternative, the associated allele, a particular
allele of a polymorphic locus, or the like is scored by single-base
extension (see e.g. U.S. Pat. No. 5,888,819). The template is first
amplified by PCR. The extension oligonucleotide is then hybridized
next to the SNP locus and the extension reaction is performed using
a thermostable polymerase such as ThermoSequenase (GE Healthcare)
in the presence of labeled ddNTPs. This reaction can therefore be
cycled several times. The identity of the labeled ddNTP
incorporated will reveal the genotype at the SNP locus. The labeled
products can be detected by means of gel electrophoresis,
fluorescence polarization (e.g. Chen et al., 1999) or by
hybridization to oligonucleotides bound to a solid support, chip,
and bead array or like. In the latter case, the extension
oligonucleotide will contain a SNP-specific zip code tag.
[0151] In yet another alternative, a SNP is scored by selective
termination of extension. The template is first amplified by PCR
and the extension oligonucleotide hybridizes in the vicinity of the
SNP locus, close to but not necessarily adjacent to it. The
extension reaction is carried out using a thermostable polymerase
such as ThermoSequenase (GE Healthcare) in the presence of a mix of
dNTPs and at least one ddNTP. The latter has to terminate the
extension at one of the allele of the interrogated SNP, but not
both such that the two alleles will generate extension products of
different sizes. The extension product can then be detected by
means of gel electrophoresis, in which case the extension products
need to be labeled, or by mass spectrometry (see e.g. Storm et al.,
2003).
[0152] In another alternative, SNPs are detected using an invasive
cleavage assay (see U.S. Pat. No. 6,090,543). There are five
oligonucleotides per SNP to interrogate but these are used in a two
step-reaction. During the primary reaction, three of the designed
oligonucleotides are first hybridized directly to the genomic DNA.
One of them is locus-specific and hybridizes up to the SNP locus
(the pairing of the 3' base at the SNP locus is not necessary).
There are two allele-specific oligonucleotides that hybridize in
tandem to the locus-specific probe but also contain a 5' flap that
is specific for each allele of the SNP. Depending upon
hybridization of the allele-specific oligonucleotides at the base
of the SNP locus, this creates a structure that is recognized by a
cleavase enzyme (U.S. Pat. No. 6,090,606) and the allele-specific
flap is released. During the secondary reaction, the flap fragments
hybridize to a specific cassette to recreate the same structure as
above except that the cleavage will release a small DNA fragment
labeled with a fluorescent dye that can be detected using regular
fluorescence detector. In the cassette, the emission of the dye is
inhibited by a quencher.
Methods to Identify Agents that Modulate the Expression of a
Nucleic Acid Encoding a Gene Involved in IBD (Ex: Crohn's
Disease).
[0153] The present invention provides methods for identifying
agents that modulate the expression of a nucleic acid encoding a
gene from Tables 2-4. Such methods may utilize any available means
of monitoring for changes in the expression level of the nucleic
acids of the invention. As used herein, an agent is said to
modulate the expression of a nucleic acid of the invention if it is
capable of up- or down-regulating expression of the nucleic acid in
a cell. Such cells can be obtained from any parts of the body such
as the GI track, colon, esophagus, stomach, rectum, jujenum, ileum,
mucosa, submucosa, cecum, rectum, scalp, blood, dermis, epidermis,
skin cells, cutaneous surfaces, intertrigious areas, genitalia,
vessels and endothelium. Some non-limiting examples of cells that
can be used are: muscle cells, nervous cells, blood and vessels
cells, dermis, epidermis and other skin cells, T cell, mast cell,
CD4+ lymphocyte, monocyte, macrophage, synovial cell, glial cell,
villous intestinal cell, neutrophilic granulocyte, eosinophilic
granulocyte, keratinocyte, lamina propria lymphocyte,
intraepithelial lymphocyte, epithelial cells and lymphocytes.
[0154] In one assay format, the expression of a nucleic acid
encoding a gene of the invention (see Tables 2-4) in a cell or
tissue sample is monitored directly by hybridization to the nucleic
acids of the invention. Cell lines or tissues are exposed to the
agent to be tested under appropriate conditions and time and total
RNA or mRNA is isolated by standard procedures such as those
disclosed in Sambrook et al., (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press).
[0155] Probes to detect differences in RNA expression levels
between cells exposed to the agent and control cells may be
prepared as described above. Hybridization conditions are modified
using known methods, such as those described by Sambrook et al.,
and Ausubel et al., as required for each probe. Hybridization of
total cellular RNA or RNA enriched for polyA RNA can be
accomplished in any available format. For instance, total cellular
RNA or RNA enriched for polyA RNA can be affixed to a solid support
and the solid support exposed to at least one probe comprising at
least one, or part of one of the sequences of the invention under
conditions in which the probe will specifically hybridize.
Alternatively, nucleic acid fragments comprising at least one, or
part of one of the sequences of the invention can be affixed to a
solid support, such as a silicon chip or a porous glass wafer. The
chip or wafer can then be exposed to total cellular RNA or polyA
RNA from a sample under conditions in which the affixed sequences
will specifically hybridize to the RNA. By examining for the
ability of a given probe to specifically hybridize to an RNA sample
from an untreated cell population and from a cell population
exposed to the agent, agents which up or down regulate expression
are identified.
Methods to Identify Agents that Modulate the Activity of a Protein
Encoded by a Gene Involved in IBD (Ex: Crohn's Disease).
[0156] The present invention provides methods for identifying
agents that modulate at least one activity of the proteins
described in Tables 2-4. Such methods may utilize any means of
monitoring or detecting the desired activity. As used herein, an
agent is said to modulate the expression of a protein of the
invention if it is capable of up- or down-regulating expression of
the protein in a cell. Such cells can be obtained from any parts of
the body such as the GI track, colon, esophagus, stomach, rectum,
jujenum, ileum, mucosa, submucosa, cecum, rectum, scalp, blood,
dermis, epidermis, skin cells, cutaneous surfaces, intertrigious
areas, genitalia, vessels and endothelium. Some non-limiting
examples of cells that can be used are: muscle cells, nervous
cells, blood and vessels cells, dermis, epidermis and other skin
cells, T cell, mast cell, CD4+ lymphocyte, monocyte, macrophage,
synovial cell, glial cell, villous intestinal cell, neutrophilic
granulocyte, eosinophilic granulocyte, keratinocyte, lamina propria
lymphocyte, intraepithelial lymphocyte, epithelial cells and
lymphocytes.
[0157] In one format, the specific activity of a protein of the
invention, normalized to a standard unit, may be assayed in a cell
population that has been exposed to the agent to be tested and
compared to an unexposed control cell population may be assayed.
Cell lines or populations are exposed to the agent to be tested
under appropriate conditions and times. Cellular lysates may be
prepared from the exposed cell line or population and a control,
unexposed cell line or population. The cellular lysates are then
analyzed with the probe.
[0158] Antibody probes can be prepared by immunizing suitable
mammalian hosts utilizing appropriate immunization protocols using
the proteins of the invention or antigen-containing fragments
thereof. To enhance immunogenicity, these proteins or fragments can
be conjugated to suitable carriers. Methods for preparing
immunogenic conjugates with carriers such as BSA, KLH or other
carrier proteins are well known in the art. In some circumstances,
direct conjugation using, for example, carbodiimide reagents may be
effective; in other instances linking reagents such as those
supplied by Pierce Chemical Co. (Rockford, Ill.) may be desirable
to provide accessibility to the hapten. The hapten peptides can be
extended at either the amino or carboxy terminus with a cysteine
residue or interspersed with cysteine residues, for example, to
facilitate linking to a carrier. Administration of the immunogens
is conducted generally by injection over a suitable time period and
with use of suitable adjuvants, as is generally understood in the
art. During the immunization schedule, titers of antibodies are
taken to determine adequacy of antibody formation. While the
polyclonal antisera produced in this way may be satisfactory for
some applications, for pharmaceutical compositions, use of
monoclonal preparations is preferred. Immortalized cell lines which
secrete the desired monoclonal antibodies may be prepared using
standard methods, see e.g., Kohler & Milstein (1992) or
modifications which affect immortalization of lymphocytes or spleen
cells, as is generally known. The immortalized cell lines secreting
the desired antibodies can be screened by immunoassay in which the
antigen is the peptide hapten, polypeptide or protein. When the
appropriate immortalized cell culture secreting the desired
antibody is identified, the cells can be cultured either in vitro
or by production in ascites fluid. The desired monoclonal
antibodies may be recovered from the culture supernatant or from
the ascites supernatant. Fragments of the monoclonal antibodies or
the polyclonal antisera which contain the immunologically
significant portion(s) can be used as antagonists, as well as the
intact antibodies. Use of immunologically reactive fragments, such
as Fab or Fab' fragments, is often preferable, especially in a
therapeutic context, as these fragments are generally less
immunogenic than the whole immunoglobulin. The antibodies or
fragments may also be produced, using current technology, by
recombinant means. Antibody regions that bind specifically to the
desired regions of the protein can also be produced in the context
of chimeras derived from multiple species. Antibody regions that
bind specifically to the desired regions of the protein can also be
produced in the context of chimeras from multiple species, for
instance, humanized antibodies. The antibody can therefore be a
humanized antibody or a human antibody, as described in U.S. Pat.
No. 5,585,089 or Riechmann et al. (1988).
[0159] Agents that are assayed in the above method can be randomly
selected or rationally selected or designed. As used herein, an
agent is said to be randomly selected when the agent is chosen
randomly without considering the specific sequences involved in the
association of the protein of the invention alone or with its
associated substrates, binding partners, etc. An example of
randomly selected agents is the use of a chemical library or a
peptide combinatorial library, or a growth broth of an organism. As
used herein, an agent is said to be rationally selected or designed
when the agent is chosen on a non-random basis which takes into
account the sequence of the target site or its conformation in
connection with the agent's action. Agents can be rationally
selected or rationally designed by utilizing the peptide sequences
that make up these sites. For example, a rationally selected
peptide agent can be a peptide whose amino acid sequence is
identical to or a derivative of any functional consensus site. The
agents of the present invention can be, as examples,
oligonucleotides, antisense polynucleotides, interfering RNA,
peptides, peptide mimetics, antibodies, antibody fragments, small
molecules, vitamin derivatives, as well as carbohydrates. Peptide
agents of the invention can be prepared using standard solid phase
(or solution phase) peptide synthesis methods, as is known in the
art. In addition, the DNA encoding these peptides may be
synthesized using commercially available oligonucleotide synthesis
instrumentation and produced recombinantly using standard
recombinant production systems. The production using solid phase
peptide synthesis is necessitated if non-gene-encoded amino acids
are to be included.
[0160] Another class of agents of the present invention includes
antibodies or fragments thereof that bind to a protein encoded by a
gene in Tables 2-4. Antibody agents can be obtained by immunization
of suitable mammalian subjects with peptides, containing as
antigenic regions, those portions of the protein intended to be
targeted by the antibodies (see section above of antibodies as
probes for standard antibody preparation methodologies).
[0161] In yet another class of agents, the present invention
includes peptide mimetics that mimic the three-dimensional
structure of the protein encoded by a gene from Tables 2-4. Such
peptide mimetics may have significant advantages over naturally
occurring peptides, including, for example: more economical
production, greater chemical stability, enhanced pharmacological
properties (half-life, absorption, potency, efficacy, etc.),
altered specificity (e.g., a broad-spectrum of biological
activities), reduced antigenicity and others. In one form, mimetics
are peptide-containing molecules that mimic elements of protein
secondary structure. The underlying rationale behind the use of
peptide mimetics is that the peptide backbone of proteins exists
chiefly to orient amino acid side chains in such a way as to
facilitate molecular interactions, such as those of antibody and
antigen. A peptide mimetic is expected to permit molecular
interactions similar to the natural molecule. In another form,
peptide analogs are commonly used in the pharmaceutical industry as
non-peptide drugs with properties analogous to those of the
template peptide. These types of non-peptide compounds are also
referred to as peptide mimetics or peptidomimetics (Fauchere, 1986;
Veber & Freidinger, 1985; Evans et al., 1987) which are usually
developed with the aid of computerized molecular modeling. Peptide
mimetics that are structurally similar to therapeutically useful
peptides may be used to produce an equivalent therapeutic or
prophylactic effect. Generally, peptide mimetics are structurally
similar to a paradigm polypeptide (i.e., a polypeptide that has a
biochemical property or pharmacological activity), but have one or
more peptide linkages optionally replaced by a linkage using
methods known in the art. Labeling of peptide mimetics usually
involves covalent attachment of one or more labels, directly or
through a spacer (e.g., an amide group), to non-interfering
position(s) on the peptide mimetic that are predicted by
quantitative structure-activity data and molecular modeling. Such
non-interfering positions generally are positions that do not form
direct contacts with the macromolecule(s) to which the peptide
mimetic binds to produce the therapeutic effect. Derivitization
(e.g., labeling) of peptide mimetics should not substantially
interfere with the desired biological or pharmacological activity
of the peptide mimetic. The use of peptide mimetics can be enhanced
through the use of combinatorial chemistry to create drug
libraries. The design of peptide mimetics can be aided by
identifying amino acid mutations that increase or decrease binding
of the protein to its binding partners. Approaches that can be used
include the yeast two hybrid method (see Chien et al., 1991) and
the phage display method. The two hybrid method detects
protein-protein interactions in yeast (Fields et al., 1989). The
phage display method detects the interaction between an immobilized
protein and a protein that is expressed on the surface of phages
such as lambda and M13 (Amberg et al., 1993; Hogrefe et al., 1993).
These methods allow positive and negative selection for
protein-protein interactions and the identification of the
sequences that determine these interactions.
[0162] Method to Diagnose Crohn's Disease
[0163] The present invention also relates to methods for diagnosing
inflammatory bowel disease or a related disease, preferably Crohn's
disease (CD), a disposition to such disease, predisposition to such
a disease and/or disease progression. In some methods, the steps
comprise contacting a target sample with (a) nucleic acid
molecule(s) or fragments thereof and comparing the concentration of
individual mRNA(s) with the concentration of the corresponding
mRNA(s) from at least one healthy donor. An aberrant (increased or
decreased) mRNA level of at least one gene from Tables 2-4, at
least 5 or 10 genes from Tables 2-4, at least 50 genes from Tables
2-4, at least 100 genes from Tables 2-4 or at least 200 genes from
Tables 2-4 determined in the sample in comparison to the control
sample is an indication of Crohn's disease or a related disease or
a disposition to such kinds of diseases. For diagnosis, samples
are, preferably, obtained from inflamed colon tissue. Samples can
also be obtained from any parts of the body such as the GI track,
colon, esophagus, stomach, rectum, jujenum, ileum, mucosa,
submucosa, cecum, rectum, scalp, blood, dermis, epidermis, skin
cells, cutaneous surfaces, intertrigious areas, genitalia, vessels
and endothelium. Some non-limiting examples of cells that can be
used are: muscle cells, nervous cells, blood and vessels cells,
dermis, epidermis and other skin cells, T cell, mast cell, CD4+
lymphocyte, monocyte, macrophage, synovial cell, glial cell,
villous intestinal cell, neutrophilic granulocyte, eosinophilic
granulocyte, keratinocyte, lamina propria lymphocyte,
intraepithelial lymphocyte, epithelial cells and lymphocytes.
[0164] For analysis of gene expression, total RNA is obtained from
cells according to standard procedures and, preferably,
reverse-transcribed. Preferably, a DNAse treatment (in order to get
rid of contaminating genomic DNA) is performed. Some non-limiting
examples of cells that can be used are: muscle cells, nervous
cells, blood and vessels cells, dermis, epidermis and other skin
cells, T cell, mast cell, CD4+ lymphocyte, monocyte, macrophage,
synovial cell, glial cell, villous intestinal cell, neutrophilic
granulocyte, eosinophilic granulocyte, keratinocyte, lamina propria
lymphocyte, intraepithelial lymphocyte, epithelial cells and
lymphocytes.
[0165] The nucleic acid molecule or fragment is typically a nucleic
acid probe for hybridization or a primer for PCR. The person
skilled in the art is in a position to design suitable nucleic
acids probes based on the information provided in the Tables of the
present invention. The target cellular component, i.e. mRNA, e.g.,
in colon tissue, may be detected directly in situ, e.g. by in situ
hybridization or it may be isolated from other cell components by
common methods known to those skilled in the art before contacting
with a probe. Detection methods include Northern blot analysis,
RNase protection, in situ methods, e.g. in situ hybridization, in
vitro amplification methods (PCR, LCR, QRNA replicase or
RNA-transcription/amplification (TAS, 3SR), reverse dot blot
disclosed in EP-B10237362) and other detection assays that are
known to those skilled in the art. Products obtained by in vitro
amplification can be detected according to established methods,
e.g. by separating the products on agarose or polyacrylamide gels
and by subsequent staining with ethidium bromide. Alternatively,
the amplified products can be detected by using labeled primers for
amplification or labeled dNTPs. Preferably, detection is based on a
microarray.
[0166] The probes (or primers) (or, alternatively, the
reverse-transcribed sample mRNAs) can be detectably labeled, for
example, with a radioisotope, a bioluminescent compound, a
chemiluminescent compound, a fluorescent compound, a metal chelate,
or an enzyme.
[0167] The present invention also relates to the use of the nucleic
acid molecules or fragments described above for the preparation of
a diagnostic composition for the diagnosis of Crohn's disease or a
disposition to such a disease.
[0168] The present invention also relates to the use of the nucleic
acid molecules of the present invention for the isolation or
development of a compound which is useful for therapy of Crohn's
disease. For example, the nucleic acid molecules of the invention
and the data obtained using said nucleic acid molecules for
diagnosis of Crohn's disease might allow for the identification of
further genes which are specifically dysregulated, and thus may be
considered as potential targets for therapeutic interventions.
[0169] The invention further provides prognostic assays that can be
used to identify subjects having or at risk of developing Crohn's
disease. In such method, a test sample is obtained from a subject
and the amount and/or concentration of the nucleic acid described
in Tables 2-4 is determined; wherein the presence of an associated
allele, a particular allele of a polymorphic locus, or the likes in
the nucleic acids sequences of this invention (see SEQ ID from
Tables 2-36) can be diagnostic for a subject having or at risk of
developing Crohn's. As used herein, a "test sample" refers to a
biological sample obtained from a subject of interest. For example,
a test sample can be a biological fluid, a cell sample, or tissue.
A biological fluid can be, but is not limited to saliva, serum,
mucus, urine, stools, spermatozoids, vaginal secretions, lymph,
amiotic liquid, pleural liquid and tears. Cells can be, but are not
limited to: muscle cells, nervous cells, blood and vessels cells,
dermis, epidermis and other skin cells, T cell, mast cell, CD4+
lymphocyte, monocyte, macrophage, synovial cell, glial cell,
villous intestinal cell, neutrophilic granulocyte, eosinophilic
granulocyte, keranocyte, lamina propria lymphocyte, intraephitelial
lymphocyte, epithelial cells and lymphocytes.
[0170] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, polypeptide, nucleic
acid such as antisense DNA or interfering RNA (RNAi), small
molecule or other drug candidate) to treat Crohn's disease.
Specifically, these assays can be used to predict whether an
individual will have an efficacious response or will experience
adverse events in response to such an agent. For example, such
methods can be used to determine whether a subject can be
effectively treated with an agent that modulates the expression
and/or activity of a gene from Tables 2-4 or the nucleic acids
described herein. In another example, an association study may be
performed to identify polymorphisms from Tables 5-36 that are
associated with a given response to the agent, e.g., an efficacious
response or the likelihood of one or more adverse events. Thus, one
embodiment of the present invention provides methods for
determining whether a subject can be effectively treated with an
agent for a disease associated with aberrant expression or activity
of a gene from Tables 2-4 in which a test sample is obtained and
nucleic acids or polypeptides from Tables 2-4 are detected (e.g.,
wherein the presence of a particular level of expression of a gene
from Tables 2-4 or a particular allelic variant of such gene, such
as polymorphisms from Tables 5-36 is diagnostic for a subject that
can be administered an agent to treat a disorder such as Crohn's
disease). In one embodiment, the method includes obtaining a sample
from a subject suspected of having Crohn's disease or an affected
individual and exposing such sample to an agent. The expression
and/or activity of the nucleic acids and/or genes of the invention
are monitored before and after treatment with such agent to assess
the effect of such agent. After analysis of the expression values,
one skilled in the art can determine whether such agent can
effectively treat such subject. In another embodiment, the method
includes obtaining a sample from a subject having or susceptible to
developing Crohn's disease and determining the allelic constitution
of polymorphisms from Tables 5-36 that is associated with a
particular response to an agent. After analysis of the allelic
constitution of the individual at the associated polymorphisms, one
skilled in the art can determine whether such agent can effectively
treat such subject.
[0171] The methods of the invention can also be used to detect
genetic alterations in a gene from Tables 2-4, thereby determining
if a subject with the lesioned gene is at risk for a disease
associated with Crohn's disease. In preferred embodiments, the
methods include detecting, in a sample of cells from the subject,
the presence or absence of a genetic alteration characterized by at
least one alteration linked to or affecting the integrity of a gene
from Tables 2-4 encoding a polypeptide or the misexpression of such
gene. For example, such genetic alterations can be detected by
ascertaining the existence of at least one of: (1) a deletion of
one or more nucleotides from a gene from Tables 2-4; (2) an
addition of one or more nucleotides to a gene from Tables 2-4; (3)
a substitution of one or more nucleotides of a gene from Tables
2-4; (4) a chromosomal rearrangement of a gene from Tables 2-4; (5)
an alteration in the level of a messenger RNA transcript of a gene
from Tables 2-4; (6) aberrant modification of a gene from Tables
2-4, such as of the methylation pattern of the genomic DNA, (7) the
presence of a non-wild type splicing pattern of a messenger RNA
transcript of a gene from Tables 2-4; (8) inappropriate
post-translational modification of a polypeptide encoded by a gene
from Tables 2-4; and (9) alternative promoter use. As described
herein, there are a large number of assay techniques known in the
art which can be used for detecting alterations in a gene from
Tables 2-4. A preferred biological sample is a peripheral blood
sample obtained by conventional means from a subject. Another
preferred biological sample is a buccal swab. Other biological
samples can be, but are not limited to, urine, stools,
spermatozoids, vaginal secretions, lymph, amiotic liquid, pleural
liquid and tears.
[0172] In certain embodiments, detection of the alteration involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al., 1988; and Nakazawa et al.,
1994), the latter of which can be particularly useful for detecting
point mutations in a gene from Tables 2-4 (see Abavaya et al.,
1995). This method can include the steps of collecting a sample of
cells from a patient, isolating nucleic acid (e.g., genomic DNA,
mRNA, or both) from the cells of the sample, contacting the nucleic
acid sample with one or more primers which specifically hybridize
to a gene from Tables 2-4 under conditions such that hybridization
and amplification of the nucleic acid from Tables 2-4 (if present)
occurs, and detecting the presence or absence of an amplification
product, or detecting the size of the amplification product and
comparing the length to a control sample. PCR and/or LCR may be
desirable to use as a preliminary amplification step in conjunction
with some of the techniques used for detecting a mutation, an
associated allele, a particular allele of a polymorphic locus, or
the like described herein.
[0173] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al., 1990), transcriptional
amplification system (Kwoh et al., 1989), Q-Beta Replicase (Lizardi
et al., 1988), isothermal amplification (e.g. Dean et al., 2002);
and Hafner et al., 2001), or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of ordinary skill in the art. These
detection schemes are especially useful for the detection of
nucleic acid molecules if such molecules are present in very low
number.
[0174] In an alternative embodiment, alterations in a gene from
Tables 2-4, from a sample cell can be identified by identifying
changes in a restriction enzyme cleavage pattern. For example,
sample and control DNA is isolated, amplified (optionally),
digested with one or more restriction endonucleases, and fragment
length sizes are determined by gel electrophoresis and compared.
Differences in fragment length sizes between sample and control DNA
indicate a mutation(s), an associated allele, a particular allele
of a polymorphic locus, or the like in the sample DNA. Moreover,
sequence specific ribozymes (see, e.g., U.S. Pat. No. 5,498,531 or
DNAzyme e.g. U.S. Pat. No. 5,807,718) can be used to score for the
presence of specific associated allele, a particular allele of a
polymorphic locus, or the likes by development or loss of a
ribozyme or DNAzyme cleavage site.
[0175] The present invention also relates to further methods for
diagnosing IBD (ex: Crohn's disease) Crohn's disease or a related
disorder, preferably Inflammation, a disposition to such disorder,
predisposition to such a disorder and/or disorder progression. In
some methods, the steps comprise contacting a target sample with
(a) nucleic molecule(s) or fragments thereof and determining the
presence or absence of a particular allele of a polymorphism that
confers a disorder-related phenotype (e.g., predisposition to such
a disorder and/or disorder progression). The presence of at least
one allele from Tables 5-36 that is associated with Crohn's disease
("associated allele"), at least 5 or 10 associated alleles from
Tables 5-36, at least 50 associated alleles from Tables 5-36 at
least 100 associated alleles from Tables 5-36, or at least 200
associated alleles from Tables 5-36 determined in the sample is an
indication of Crohn's disease or a related disorder, a disposition
or predisposition to such kinds of disorders, or a prognosis for
such disorder progression. Samples may be obtained from any parts
of the body such as the GI track, colon, esophagus, stomach,
rectum, jujenum, ileum, mucosa, submucosa, cecum, rectum, scalp,
blood, dermis, epidermis, skin cells, cutaneous surfaces,
intertrigious areas, genitalia, vessels and endothelium. Some
non-limiting examples of cells that can be used are: muscle cells,
nervous cells, blood and vessels cells, dermis, epidermis and other
skin cells, T cell, mast cell, CD4+ lymphocyte, monocyte,
macrophage, synovial cell, glial cell, villous intestinal cell,
neutrophilic granulocyte, eosinophilic granulocyte, keratinocyte,
lamina propria lymphocyte, intraepithelial lymphocyte, epithelial
cells and lymphocytes.
[0176] In other embodiments, alterations in a gene from Tables 2-4
can be identified by hybridizing sample and control nucleic acids,
e.g., DNA or RNA, to high density arrays or bead arrays containing
tens to thousands of oligonucleotide probes (Cronin et al., 1996;
Kozal et al., 1996). For example, alterations in a gene from Tables
2-4 can be identified in two dimensional arrays containing
light-generated DNA probes as described in Cronin et al., (1996).
Briefly, a first hybridization array of probes can be used to scan
through long stretches of DNA in a sample and control to identify
base changes between the sequences by making linear arrays of
sequential overlapping probes. This step allows the identification
of point mutations, associated alleles, particular alleles of a
polymorphic locus, or the like. This step is followed by a second
hybridization array that allows the characterization of specific
mutations by using smaller, specialized probe arrays complementary
to all variants, mutations, alleles detected. Each mutation array
is composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant gene.
[0177] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence a gene
from Tables 2-4 and detect an associated allele, a particular
allele of a polymorphic locus, or the like by comparing the
sequence of the sample gene from Tables 2-4 with the corresponding
wild-type (control) sequence. Examples of sequencing reactions
include those based on techniques developed by Maxam and Gilbert
(1977) or Sanger (1977). It is also contemplated that any of a
variety of automated sequencing procedures can be utilized when
performing the diagnostic assays (Bio/Techniques 19:448, 1995)
including sequencing by mass spectrometry (see, e.g. PCT
International Publication No. WO 94/16101; Cohen et al., 1996; and
Griffin et al. 1993), real-time pyrophosphate sequencing method
(Ronaghi et al., 1998; and Permutt et al., 2001) and sequencing by
hybridization (see e.g. Drmanac et al., 2002).
[0178] Other methods of detecting an associated allele, a
particular allele of a polymorphic locus, or the likes in a gene
from Tables 2-4 include methods in which protection from cleavage
agents is used to detect mismatched bases in RNA/RNA, DNA/DNA or
RNA/DNA heteroduplexes (Myers et al., 1985). In general, the
technique of "mismatch cleavage" starts by providing heteroduplexes
formed by hybridizing (labeled) RNA or DNA containing the wild-type
gene sequence from Tables 2-4 with potentially mutant RNA or DNA
obtained from a tissue sample. The double-stranded duplexes are
treated with an agent that cleaves single-stranded regions of the
duplex such as which will exist due to basepair mismatches between
the control and sample strands. For instance, RNA/DNA duplexes can
be treated with RNase and DNA/DNA hybrids treated with S1 nuclease
to enzymatically digest the mismatched regions. In other
embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with
hydroxylamine or osmium tetroxide and with piperidine in order to
digest mismatched regions. After digestion of the mismatched
regions, the resulting material is then separated by size on
denaturing polyacrylamide gels to determine the site of an
associated allele, a particular allele of a polymorphic locus, or
the like (see, for example, Cotton et al., 1988; Saleeba et al.,
1992). In a preferred embodiment, the control DNA or RNA can be
labeled for detection, as described herein.
[0179] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point an associated
allele, a particular allele of a polymorphic locus, or the likes in
a gene from Tables 2-4 cDNAs obtained from samples of cells. For
example, the mutY enzyme of E. coli cleaves A at G/A mismatches
(Hsu et al., 1994). Other examples include, but are not limited to,
the MutHLS enzyme complex of E. coli (Smith and Modrich., 1996) and
Cel 1 from the celery (Kulinski et al., 2000) both cleave the DNA
at various mismatches. According to an exemplary embodiment, a
probe based on a gene sequence from Tables 2-4 is hybridized to a
cDNA or other DNA product from a test cell or cells. The duplex is
treated with a DNA mismatch repair enzyme, and the cleavage
products, if any, can be detected using electrophoresis protocols
or the like. See, for example, U.S. Pat. No. 5,459,039.
Alternatively, the screen can be performed in vivo following the
insertion of the heteroduplexes in an appropriate vector. The whole
procedure is known to those ordinary skilled in the art and is
referred to as mismatch repair detection (see e.g. Fakhrai-Rad et
al., 2004).
[0180] In other embodiments, alterations in electrophoretic
mobility can be used to identify an associated allele, a particular
allele of a polymorphic locus, or the likes in genes from Tables
2-4. For example, single strand conformation polymorphism (SSCP)
analysis can be used to detect differences in electrophoretic
mobility between mutant and wild type nucleic acids (Orita et al.,
1993; see also Cotton, 1993; and Hayashi et al., 1992).
Single-stranded DNA fragments of sample and control nucleic acids
from Tables 2-4 will be denatured and allowed to renature. The
secondary structure of single-stranded nucleic acids varies
according to sequence; the resulting alteration in electrophoretic
mobility enables the detection of even a single base change. The
DNA fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the method utilizes
heteroduplex analysis to separate double stranded heteroduplex
molecules on the basis of changes in electrophoretic mobility (Kee
et al., 1991).
[0181] In yet another embodiment, the movement of mutant or
wild-type fragments in a polyacrylamide gel containing a gradient
of denaturant is assayed using denaturing gradient gel
electrophoresis (DGGE) (Myers et al., 1985). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum et al., 1987). In another
embodiment, the mutant fragment is detected using denaturing HPLC
(see e.g. Hoogendoorn et al., 2000).
[0182] Examples of other techniques for detecting point mutations,
an associated allele, a particular allele of a polymorphic locus,
or the like include, but are not limited to, selective
oligonucleotide hybridization, selective amplification, selective
primer extension, selective ligation, single-base extension,
selective termination of extension or invasive cleavage assay. For
example, oligonucleotide primers may be prepared in which the known
associated allele, particular allele of a polymorphic locus, or the
like is placed centrally and then hybridized to target DNA under
conditions which permit hybridization only if a perfect match is
found (Saiki et al., 1986; Saiki et al., 1989). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA of a number of different associated alleles, a particular
allele of a polymorphic locus, or the likes where the
oligonucleotides are attached to the hybridizing membrane and
hybridized with labeled target DNA. Alternatively, the
amplification, the allele-specific hybridization and the detection
can be done in a single assay following the principle of the 5'
nuclease assay (e.g. see Livak et al., 1995). For example, the
associated allele, a particular allele of a polymorphic locus, or
the like locus is amplified by PCR in the presence of both
allele-specific oligonucleotides, each specific for one or the
other allele. Each probe has a different fluorescent dye at the 5'
end and a quencher at the 3' end. During PCR, if one or the other
or both allele-specific oligonucleotides are hybridized to the
template, the Taq polymerase via its 5' exonuclease activity will
release the corresponding dyes. The latter will thus reveal the
genotype of the amplified product.
[0183] The hybridization may also be carried out with a temperature
gradient following the principle of dynamic allele-specific
hybridization or like (e.g. Jobs et al., 2003; and Bourgeois and
Labuda, 2004). For example, the hybridization is done using one of
the two allele-specific oligonucleotides labeled with a fluorescent
dye, an intercalating quencher under a gradually increasing
temperature. At low temperature, the probe is hybridized to both
the mismatched and full-matched template. The probe melts at a
lower temperature when hybridized to the template with a mismatch.
The release of the probe is captured by an emission of the
fluorescent dye, away from the quencher. The probe melts at a
higher temperature when hybridized to the template with no
mismatch. The temperature-dependent fluorescence signals therefore
indicate the absence or presence of the associated allele,
particular allele of a polymorphic locus, or the like (e.g. Jobs et
al. supra). Alternatively, the hybridization is done under a
gradually decreasing temperature. In this case, both
allele-specific oligonucleotides are hybridized to the template
competitively. At high temperature none of the two probes is
hybridized. Once the optimal temperature of the full-matched probe
is reached, it hybridizes and leaves no target for the mismatched
probe. In the latter case, if the allele-specific probes are
differently labeled, then they are hybridized to a single
PCR-amplified target. If the probes are labeled with the same dye,
then the probe cocktail is hybridized twice to identical templates
with only one labeled probe, different in the two cocktails, in the
presence of the unlabeled competitive probe.
[0184] Alternatively, allele specific amplification technology that
depends on selective PCR amplification may be used in conjunction
with the present invention. Oligonucleotides used as primers for
specific amplification may carry the associated allele, particular
allele of a polymorphic locus, or the like of interest in the
center of the molecule, so that amplification depends on
differential hybridization (Gibbs et al., 1989) or at the extreme
3' end of one primer where, under appropriate conditions, mismatch
can prevent, or reduce polymerase extension (Prossner, 1993). In
addition it may be desirable to introduce a novel restriction site
in the region of the associated allele, particular allele of a
polymorphic locus, or the like to create cleavage-based detection
(Gasparini et al., 1992). It is anticipated that in certain
embodiments amplification may also be performed using Taq ligase
for amplification (Barany, 1991). In such cases, ligation will
occur only if there is a perfect match at the 3' end of the 5'
sequence making it possible to detect the presence of a known
associated allele, a particular allele of a polymorphic locus, or
the like at a specific site by looking for the presence or absence
of amplification. The products of such an oligonucleotide ligation
assay can also be detected by means of gel electrophoresis.
Furthermore, the oligonucleotides may contain universal tags used
in PCR amplification and zip code tags that are different for each
allele. The zip code tags are used to isolate a specific, labeled
oligonucleotide that may contain a mobility modifier (e.g. Grossman
et al., 1994).
[0185] In yet another alternative, allele-specific elongation
followed by ligation will form a template for PCR amplification. In
such cases, elongation will occur only if there is a perfect match
at the 3' end of the allele-specific oligonucleotide using a DNA
polymerase. This reaction is performed directly on the genomic DNA
and the extension/ligation products are amplified by PCR. To this
end, the oligonucleotides contain universal tags allowing
amplification at a high multiplex level and a zip code for SNP
identification. The PCR tags are designed in such a way that the
two alleles of a SNP are amplified by different forward primers,
each having a different dye. The zip code tags are the same for
both alleles of a given SNP and they are used for hybridization of
the PCR-amplified products to oligonucleotides bound to a solid
support, chip, bead array or like. For an example of the procedure,
see Fan et al. (Cold Spring Harbor Symposia on Quantitative
Biology, Vol. LXVIII, pp. 69-78, 2003).
[0186] Another alternative includes the single-base
extension/ligation assay using a molecular inversion probe,
consisting of a single, long oligonucleotide (see e.g. Hardenbol et
al., 2003). In such an embodiment, the oligonucleotide hybridizes
on both sides of the SNP locus directly on the genomic DNA, leaving
a one-base gap at the SNP locus. The gap-filling, one-base
extension/ligation is performed in four tubes, each having a
different dNTP. Following this reaction, the oligonucleotide is
circularized whereas unreactive, linear oligonucleotides are
degraded using an exonulease such as exonuclease I of E. coli. The
circular oligonucleotides are then linearized and the products are
amplified and labeled using universal tags on the oligonucleotides.
The original oligonucleotide also contains a SNP-specific zip code
allowing hybridization to oligonucleotides bound to a solid
support, chip, bead array or the like. This reaction can be
performed at a highly multiplexed level.
[0187] In another alternative, the associated allele, particular
allele of a polymorphic locus, or the like is scored by single-base
extension (see e.g. U.S. Pat. No. 5,888,819). The template is first
amplified by PCR. The extension oligonucleotide is then hybridized
next to the SNP locus and the extension reaction is performed using
a thermostable polymerase such as ThermoSequenase (GE Healthcare)
in the presence of labeled ddNTPs. This reaction can therefore be
cycled several times. The identity of the labeled ddNTP
incorporated will reveal the genotype at the SNP locus. The labeled
products can be detected by means of gel electrophoresis,
fluorescence polarization (e.g. Chen et al., 1999) or by
hybridization to oligonucleotides bound to a solid support, chip,
bead array or the like. In the latter case, the extension
oligonucleotide will contain a SNP-specific zip code tag.
[0188] In yet another alternative, the variant is scored by
selective termination of extension. The template is first amplified
by PCR and the extension oligonucleotide hybridizes in vicinity to
the SNP locus, close to but not necessarily adjacent to it. The
extension reaction is carried out using a thermostable polymerase
such as ThermoSequenase (GE Healthcare) in the presence of a mix of
dNTPs and at least one ddNTP. The latter has to terminate the
extension at one of the alleles of the interrogated SNP, but not
both such that the two alleles will generate extension products of
different sizes. The extension product can then be detected by
means of gel electrophoresis, in which case the extension products
need to be labeled, or by mass spectrometry (see e.g. Storm et al.,
2003).
[0189] In another alternative, the associated allele, particular
allele of a polymorphic locus, or the like is detected using an
invasive cleavage assay (see U.S. Pat. No. 6,090,543). There are
five oligonucleotides per SNP to interrogate but these are used in
a two step-reaction. During the primary reaction, three of the
designed oligonucleotides are first hybridized directly to the
genomic DNA. One of them is locus-specific and hybridizes up to the
SNP locus (the pairing of the 3' base at the SNP locus is not
necessary). There are two allele-specific oligonucleotides that
hybridize in tandem to the locus-specific probe but also contain a
5' flap that is specific for each allele of the SNP. Depending upon
hybridization of the allele-specific oligonucleotides at the base
of the SNP locus, this creates a structure that is recognized by a
cleavase enzyme (U.S. Pat. No. 6,090,606) and the allele-specific
flap is released. During the secondary reaction, the flap fragments
hybridize to a specific cassette to recreate the same structure as
above except that the cleavage will release a small DNA fragment
labeled with a fluorescent dye that can be detected using regular
fluorescence detector. In the cassette, the emission of the dye is
inhibited by a quencher.
[0190] Other types of markers can also be used for diagnostic
purposes. For example, microsatellites can also be useful to detect
the genetic predisposition of an individual to a given disorder.
Microsatellites consist of short sequence motifs of one or a few
nucleotides repeated in tandem. The most common motifs are
polynucleotide runs, dinucleotide repeats (particularly the CA
repeats) and trinucleotide repeats. However, other types of repeats
can also be used. The microsatellites are very useful for genetic
mapping because they are highly polymorphic in their length.
Microsatellite markers can be typed by various means, including but
not limited to DNA fragment sizing, oligonucleotide ligation assay
and mass spectrometry. For example, the locus of the microsatellite
is amplified by PCR and the size of the PCR fragment will be
directly correlated to the length of the microsatellite repeat. The
size of the PCR fragment can be detected by regular means of gel
electrophoresis. The fragment can be labeled internally during PCR
or by using end-labeled oligonucleotides in the PCR reaction (e.g.
Mansfield et al., 1996). Alternatively, the size of the PCR
fragment is determined by mass spectrometry. In such a case,
however, the flanking sequences need to be eliminated. This can be
achieved by ribozyme cleavage of an RNA transcript of the
microsatellite repeat (Krebs et al., 2001). For example, the
microsatellite locus is amplified using oligonucleotides that
include a T7 promoter on one end and a ribozyme motif on the other
end. Transcription of the amplified fragments will yield an RNA
substrate for the ribozyme, releasing small RNA fragments that
contain the repeated region. The size of the latter is determined
by mass spectrometry. Alternatively, the flanking sequences are
specifically degraded. This is achieved by replacing the dTTP in
the PCR reaction by dUTP. The dUTP nucleosides are then removed by
uracyl DNA glycosylases and the resulting abasic sites are cleaved
by either abasic endonucleases such as human AP endonuclease or
chemical agents such as piperidine. Bases can also be modified
post-PCR by chemical agents such as dimethyl sulfate and then
cleaved by other chemical agents such as piperidine (see e.g. Maxam
and Gilbert, 1977; U.S. Pat. No. 5,869,242; and U.S. patent pending
Ser. No. 60/335,068).
[0191] In another alternative, an oligonucleotide ligation assay
can be performed. The microsatellite locus is first amplified by
PCR. Then, different oligonucleotides can be submitted to ligation
at the center of the repeat with a set of oligonucleotides covering
all the possible lengths of the marker at a given locus (Zirvi et
al., 1999). Another example of design of an oligonucleotide assay
comprises the ligation of three oligonucleotides; a 5'
oligonucleotide hybridizing to the 5' flanking sequence, a repeat
oligonucleotide of the length of the shortest allele of the marker
hybridizing to the repeated region and a set of 3' oligonucleotides
covering all the existing alleles hybridizing to the 3' flanking
sequence and a portion of the repeated region for all the alleles
longer than the shortest one. For the shortest allele, the 3'
oligonucleotide exclusively hybridizes to the 3' flanking sequence
(U.S. Pat. No. 6,479,244).
[0192] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid selected from the SEQ ID of Tables 2-36, or
antibody reagent described herein, which may be conveniently used,
for example, in a clinical setting to diagnose patient exhibiting
symptoms or a family history of a disorder or disorder involving
abnormal activity of genes from Tables 2-4.
[0193] Method to Treat an Animal Suspected of Having IBD (Ex:
Crohn's Disease)
[0194] The present invention provides methods of treating a disease
associated with IBD (ex: Crohn's disease) by expressing in vivo the
nucleic acids of at least one gene from Tables 2-4. These nucleic
acids can be inserted into any of a number of well-known vectors
for the transfection of target cells and organisms as described
below. The nucleic acids are transfected into cells, ex vivo or in
vivo, through the interaction of the vector and the target cell.
The nucleic acids encoding a gene from Tables 2-4, under the
control of a promoter, then express the encoded protein, thereby
mitigating the effects of absent, partial inactivation, or abnormal
expression of a gene from Tables 2-4.
[0195] Such gene therapy procedures have been used to correct
acquired and inherited genetic defects, cancer, and viral infection
in a number of contexts. The ability to express artificial genes in
humans facilitates the prevention and/or cure of many important
human disorders, including many disorders which are not amenable to
treatment by other therapies (for a review of gene therapy
procedures, see Anderson, 1992; Nabel & Felgner, 1993; Mitani
& Caskey, 1993; Mulligan, 1993; Dillon, 1993; Miller, 1992; Van
Brunt, 1998; Vigne, 1995; Kremer & Perricaudet 1995; Doerfler
& Bohm 1995; and Yu et al., 1994).
[0196] Delivery of the gene or genetic material into the cell is
the first critical step in gene therapy treatment of a disorder. A
large number of delivery methods are well known to those of skill
in the art. Preferably, the nucleic acids are administered for in
vivo or ex vivo gene therapy uses. Non-viral vector delivery
systems include DNA plasmids, naked nucleic acid, and nucleic acid
complexed with a delivery vehicle such as a liposome. Viral vector
delivery systems include DNA and RNA viruses, which have either
episomal or integrated genomes after delivery to the cell. For a
review of gene therapy procedures, see the references included in
the above section.
[0197] The use of RNA or DNA based viral systems for the delivery
of nucleic acids take advantage of highly evolved processes for
targeting a virus to specific cells in the body and trafficking the
viral payload to the nucleus. Viral vectors can be administered
directly to patients (in vivo) or they can be used to treat cells
in vitro and the modified cells are administered to patients (ex
vivo). Conventional viral based systems for the delivery of nucleic
acids could include retroviral, lentivirus, adenoviral,
adeno-associated and herpes simplex virus vectors for gene
transfer. Viral vectors are currently the most efficient and
versatile method of gene transfer in target cells and tissues.
Integration in the host genome is possible with the retrovirus,
lentivirus, and adeno-associated virus gene transfer methods, often
resulting in long term expression of the inserted transgene.
Additionally, high transduction efficiencies have been observed in
many different cell types and target tissues.
[0198] The tropism of a retrovirus can be altered by incorporating
foreign envelope proteins, expanding the potential target
population of target cells. Lentiviral vectors are retroviral
vectors that are able to transduce or infect non-dividing cells and
typically produce high viral titers. Selection of a retroviral gene
transfer system would therefore depend on the target tissue.
Retroviral vectors are comprised of cis-acting long terminal
repeats with packaging capacity for up to 6-10 kb of foreign
sequence. The minimum cis-acting LTRs are sufficient for
replication and packaging of the vectors, which are then used to
integrate the therapeutic gene into the target cell to provide
permanent transgene expression. Widely used retroviral vectors
include those based upon murine leukemia virus (MuLV), gibbon ape
leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human
immuno deficiency virus (HIV), and combinations thereof (see, e.g.,
Buchscher et al., 1992; Johann et al., 1992; Sommerfelt et al.,
1990; Wilson et al., 1989; Miller et al., 1999; and
PCT/US94/05700).
[0199] In applications where transient expression of the nucleic
acid is preferred, adenoviral based systems are typically used.
Adenoviral based vectors are capable of very high transduction
efficiency in many cell types and do not require cell division.
With such vectors, high titer and levels of expression have been
obtained. This vector can be produced in large quantities in a
relatively simple system. Adeno-associated virus ("AAV") vectors
are also used to transduce cells with target nucleic acids, e.g.,
in the in vitro production of nucleic acids and peptides, and for
in vivo and ex vivo gene therapy procedures (see, e.g., West et
al., 1987; U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, 1994;
Muzyczka, 1994). Construction of recombinant AAV vectors is
described in a number of publications, including U.S. Pat. No.
5,173,414; Tratschin et al., 1985; Tratschin, et al., 1984;
Hermonat & Muzyczka, 1984; and Samulski et al., 1989.
[0200] In particular, numerous viral vector approaches are
currently available for gene transfer in clinical trials, with
retroviral vectors by far the most frequently used system. All of
these viral vectors utilize approaches that involve complementation
of defective vectors by genes inserted into helper cell lines to
generate the transducing agent. pLASN and MFG-S are examples are
retroviral vectors that have been used in clinical trials (Dunbar
et al., 1995; Kohn et al., 1995; Malech et al., 1997). PA317/pLASN
was the first therapeutic vector used in a gene therapy trial
(Blaese et al., 1995). Transduction efficiencies of 50% or greater
have been observed for MFG-S packaged vectors (Ellem et al., 1997;
and Dranoff et al., 1997).
[0201] Recombinant adeno-associated virus vectors (rAAV) are a
promising alternative gene delivery systems based on the defective
and nonpathogenic parvovirus adeno-associated type 2 virus. All
vectors are derived from a plasmid that retains only the AAV 145 bp
inverted terminal repeats flanking the transgene expression
cassette. Efficient gene transfer and stable transgene delivery due
to integration into the genomes of the transduced cell are key
features for this vector system (Wagner et al., 1998, Kearns et
al., 1996).
[0202] Replication-deficient recombinant adenoviral vectors (Ad)
are predominantly used in transient expression gene therapy;
because they can be produced at high titer and they readily infect
a number of different cell types. Most adenovirus vectors are
engineered such that a transgene replaces the Ad E1a, E1b, and E3
genes; subsequently the replication defector vector is propagated
in human 293 cells that supply the deleted gene function in trans.
Ad vectors can transduce multiple types of tissues in vivo,
including nondividing, differentiated cells such as those found in
the liver, kidney and muscle tissues. Conventional Ad vectors have
a large carrying capacity. An example of the use of an Ad vector in
a clinical trial involved polynucleotide therapy for antitumor
immunization with intramuscular injection (Sterman et al., 1998).
Additional examples of the use of adenovirus vectors for gene
transfer in clinical trials include Rosenecker et al., 1996;
Sterman et al., 1998; Welsh et al., 1995; Alvarez et al., 1997;
Topf et al., 1998.
[0203] Packaging cells are used to form virus particles that are
capable of infecting a host cell. Such cells include 293 cells,
which package adenovirus, and .psi.2 cells or PA317 cells, which
package retrovirus. Viral vectors used in gene therapy are usually
generated by a producer cell line that packages a nucleic acid
vector into a viral particle. The vectors typically contain the
minimal viral sequences required for packaging and subsequent
integration into a host, other viral sequences being replaced by an
expression cassette for the protein to be expressed. The missing
viral functions are supplied in trans by the packaging cell line.
For example, AAV vectors used in gene therapy typically only
possess ITR sequences from the AAV genome which are required for
packaging and integration into the host genome. Viral DNA is
packaged in a cell line, which contains a helper plasmid encoding
the other AAV genes, namely rep and cap, but lacking ITR sequences.
The cell line is also infected with adenovirus as a helper. The
helper virus promotes replication of the AAV vector and expression
of AAV genes from the helper plasmid. The helper plasmid is not
packaged in significant amounts due to a lack of ITR sequences.
Contamination with adenovirus can be reduced by, e.g., heat
treatment to which adenovirus is more sensitive than AAV.
[0204] In many gene therapy applications, it is desirable that the
gene therapy vector be delivered with a high degree of specificity
to a particular tissue type. A viral vector is typically modified
to have specificity for a given cell type by expressing a ligand as
a fusion protein with a viral coat protein on the viruses outer
surface. The ligand is chosen to have affinity for a receptor known
to be present on the cell type of interest. For example, Han et
al., 1995, reported that Moloney murine leukemia virus can be
modified to express human heregulin fused to gp70, and the
recombinant virus infects certain human breast cancer cells
expressing human epidermal growth factor receptor. This principle
can be extended to other pairs of viruses expressing a ligand
fusion protein and target cells expressing a receptor. For example,
filamentous phage can be engineered to display antibody fragments
(e.g., Fab or Fv) having specific binding affinity for virtually
any chosen cellular receptor. Although the above description
applies primarily to viral vectors, the same principles can be
applied to nonviral vectors. Such vectors can be engineered to
contain specific uptake sequences thought to favor uptake by
specific target cells.
[0205] Gene therapy vectors can be delivered in vivo by
administration to an individual patient, typically by systemic
administration (e.g., intravenous, intraperitoneal, intramuscular,
subdermal, or intracranial infusion) or topical application.
Alternatively, vectors can be delivered to cells ex vivo, such as
cells explanted from an individual patient (e.g., lymphocytes, bone
marrow aspirates, and tissue biopsy) or universal donor
hematopoietic stem cells, followed by reimplantation of the cells
into a patient, usually after selection for cells which have
incorporated the vector.
[0206] Ex vivo cell transfection for diagnostics, research, or for
gene therapy (e.g., via re-infusion of the transfected cells into
the host organism) is well known to those of skill in the art. In a
preferred embodiment, cells are isolated from the subject organism,
transfected with a nucleic acid (gene or cDNA), and re-infused back
into the subject organism (e.g., patient). Various cell types
suitable for ex vivo transfection are well known to those of skill
in the art (see, e.g., Freshney et al., 1994; and the references
cited therein for a discussion of how to isolate and culture cells
from patients).
[0207] In one embodiment, stem cells are used in ex vivo procedures
for cell transfection and gene therapy. The advantage to using stem
cells is that they can be differentiated into other cell types in
vitro, or can be introduced into a mammal (such as the donor of the
cells) where they will engraft in the bone marrow. Methods for
differentiating CD34+ cells in vitro into clinically important
immune cell types using cytokines such a GM-CSF, IFN-.gamma. and
TNF-.alpha., are known (see Inaba et al., 1992).
[0208] Stem cells are isolated for transduction and differentiation
using known methods. For example, stem cells are isolated from bone
marrow cells by panning the bone marrow cells with antibodies which
bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+(panB
cells), GR-1 (granulocytes), and lad (differentiated antigen
presenting cells).
[0209] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)
containing therapeutic nucleic acids can be also administered
directly to the organism for transduction of cells in vivo.
Alternatively, naked DNA can be administered.
[0210] Administration is by any of the routes normally used for
introducing a molecule into ultimate contact with blood or tissue
cells, as described above. The nucleic acids from Tables 2-4 are
administered in any suitable manner, preferably with the
pharmaceutically acceptable carriers described above. Suitable
methods of administering such nucleic acids are available and well
known to those of skill in the art, and, although more than one
route can be used to administer a particular composition, a
particular route can often provide a more immediate and more
effective reaction than another route (see Samulski et al., 1989).
The present invention is not limited to any method of administering
such nucleic acids, but preferentially uses the methods described
herein.
[0211] The present invention further provides other methods of
treating IBD (ex: Crohn's disease or UC) such as administering to
an individual having IBD (ex: Crohn's disease or UC) an effective
amount of an agent that regulates the expression, activity or
physical state of at least one gene from Tables 2-4. An "effective
amount" of an agent is an amount that modulates a level of
expression or activity of a gene from Tables 2-4, in a cell in the
individual at least about 10%, at least about 20%, at least about
30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80% or more, compared to a level of
the respective gene from Tables 2-4 in a cell in the individual in
the absence of the compound. The preventive or therapeutic agents
of the present invention may be administered, either orally or
parenterally, systemically or locally. For example, intravenous
injection such as drip infusion, intramuscular injection,
intraperitoneal injection, subcutaneous injection, suppositories,
intestinal lavage, oral enteric coated tablets, and the like can be
selected, and the method of administration may be chosen, as
appropriate, depending on the age and the conditions of the
patient. The effective dosage is chosen from the range of 0.01 mg
to 100 mg per kg of body weight per administration. Alternatively,
the dosage in the range of 1 to 1000 mg, preferably 5 to 50 mg per
patient may be chosen. The therapeutic efficacy of the treatment
may be monitored by observing various parts of the GI tract, by
endoscopy, barium, colonoscopy, or any other monitoring methods
known in the art. Other ways of monitoring efficacy can be, but are
not limited to monitoring inflammatory conditions involving the
upper gastrointestinal tract such as monitoring the amelioration on
the esophageal discomfort, decrease in pain, improved swallowing,
reduced chest pain, decreased heartburn, decreased regurgitation of
solids or liquids after swallowing or eating, decrease in vomiting,
or improvement in weight gain or improvement in vitality.
[0212] The present invention further provides a method of treating
an individual clinically diagnosed with IBD (ex: Crohn's disease or
UC). The methods generally comprises analyzing a biological sample
that includes a cell, in some cases, a GI track cell, from an
individual clinically diagnosed with IBD (ex: Crohn's disease or
UC) for the presence of modified levels of expression of at least 1
gene, at least 10 genes, at least 50 genes, at least 100 genes, or
at least 200 genes from Tables 2-4. A treatment plan that is most
effective for individuals clinically diagnosed as having a
condition associated with Crohn's disease is then selected on the
basis of the detected expression of such genes in a cell. Treatment
may include administering a composition that includes an agent that
modulates the expression or activity of a protein from Tables 2-4
in the cell. Information obtained as described in the methods above
can also be used to predict the response of the individual to a
particular agent. Thus, the invention further provides a method for
predicting a patient's likelihood to respond to a drug treatment
for a condition associated with IBD (ex: Crohn's disease or UC),
comprising determining whether modified levels of a gene from
Tables 2-4 is present in a cell, wherein the presence of protein is
predictive of the patient's likelihood to respond to a drug
treatment for the condition. Examples of the prevention or
improvement of symptoms accompanied by IBD (ex: Crohn's disease or
UC) that can monitored for effectiveness include prevention or
improvement of diarrhea, prevention or improvement of weight loss,
inhibition of bowel tissue edema, inhibition of cell infiltration,
inhibition of surviving period shortening, and the like, and as a
result, a preventing or improving agent for diarrhea, a preventing
or improving agent for weight loss, an inhibitor for bowel tissues
edema, an inhibitor for cell infiltration, an inhibitor for
surviving period shortening, and the like can be identified.
[0213] The invention also provides a method of predicting a
response to therapy in a subject having IBD (ex: Crohn's disease or
UC) by determining the presence or absence in the subject of one or
more markers associated with IBD (ex: Crohn's disease or UC)
described in Tables 5-36, diagnosing the subject in which the one
or more markers are present as having IBD (ex: Crohn's disease or
UC), and predicting a response to a therapy based on the diagnosis
e.g., response to therapy may include an efficacious response
and/or one or more adverse events. The invention also provides a
method of optimizing therapy in a subject having IBD (ex: Crohn's
disease or UC) by determining the presence or absence in the
subject of one or more markers associated with a clinical subtype
of IBD (ex: Crohn's disease or UC), diagnosing the subject in which
the one or more markers are present as having a particular clinical
subtype of IBD (ex: Crohn's disease or UC), and treating the
subject having a particular clinical subtype of IBD (ex: Crohn's
disease or UC) based on the diagnosis. As an example, treatment for
the fibrostenotic subtype of IBD (ex: Crohn's disease or UC)
currently includes surgical removal of the affected, structured
part of the bowel.
[0214] Thus, while there are a number of treatments for IBD (ex:
Crohn's disease or UC) currently available, they all are
accompanied by various side effects, high costs, and long
complicated treatment protocols, which are often not available and
effective in a large number of individuals. Accordingly, there
remains a need in the art for more effective and otherwise improved
methods for treating and preventing IBD (ex: Crohn's disease or
UC). Thus, there is a continuing need in the medical arts for
genetic markers of IBD (ex: Crohn's disease or UC) and guidance for
the use of such markers. The present invention fulfills this need
and provides further related advantages.
EXAMPLES
Example 1
Identification of Cases and Controls
[0215] All individuals were sampled from the Quebec founder
population (QFP). Membership in the founder population was defined
as having four grandparents with French Canadian family names who
were born in the Province of Quebec, Canada or in adjacent areas of
the Provinces of New Brunswick and Ontario or in New England or New
York State. The Quebec population is characterized both by extended
LD and by decreased genetic heterogeneity. The increased extent of
LD allows the detection of disease associated genes using a
reasonable marker density, while still allowing the increased
meiotic resolution of population-based mapping. The specific
combination of age in generations, optimal number of founders and
large present population size makes the QFP optimal for LD-based
gene mapping.
[0216] Patient inclusion criteria for the study include diagnosis
for Crohn's disease by any one of the following: a colonoscopy, a
radiological examination with barium, an abdominal surgical
operation or a biopsy or a surgical specimen. The colonoscopy
diagnosis consists of observing linear, deep or serpentigenous
ulcers, pseudopolyps, or skip areas. The barium radiological
examination consists of the detection of strictures, ulcerations
and string signs by observing the barium enema and the small bowel
followed through an NMRI series.
[0217] Patients that were diagnosed with ulcerative colitis,
infectious colitis or other intestinal diseases were excluded from
the study. All human sampling was subject to ethical review
procedures.
[0218] All enrolled QFP subjects (patients and controls) provided a
30 ml blood sample (3 barcoded tubes of 10 ml). Samples were
processed immediately upon arrival at Genizon's laboratory. All
samples were scanned and logged into a LabVantage Laboratory
Information Management System (LIMS), which served as a hub between
the clinical data management system and the genetic analysis
system. Following centrifugation, the buffy coat containing the
white blood cells was isolated from each tube. Genomic DNA was
extracted from the buffy coat from one of the tubes, and stored at
4.degree. C. until required for genotyping. DNA extraction was
performed with a commercial kit using a guanidine hydrochloride
based method (FlexiGene, Qiagen) according to the manufacturer's
instructions. The extraction method yielded high molecular weight
DNA, and the quality of every DNA sample was verified by agarose
gel electrophoresis. Genomic DNA appeared on the gel as a large
band of very high molecular weight. The remaining two buffy coats
were stored at -80.degree. C. as backups.
[0219] The QFP samples were collected as family trios consisting of
Crohn's disease subjects and two first degree relatives. Of the 500
trios, 477 were Parent, Parent, Child (PPC) trios; the remainders
were Parent, Child, Child (PCC) trios. Only the PPC trios were used
for the analysis reported here because they produced equal numbers
of more accurately estimated case and control haplotypes than the
PCC trios. 382 trios were used in the genome wide scan. One member
of each trio was affected with Crohn's disease. For the 382 trios
used in the genome wide scan, these included 189 daughters, 90
sons, 54 mothers and 49 fathers. When a child was the affected
member of the trio, the two non-transmitted parental chromosomes
(one from each parent) were used as controls, when one of the
parents was affected, that person's spouse provided the control
chromosomes. The recruitment of trios allowed a more precise
determination of long extended haplotypes.
Example 2
Genome Wide Association
[0220] Genotyping was performed using Perlegen's
ultra-high-throughput platform. Marker loci were amplified by PCR
and hybridized to wafers containing arrays of oligonucleotides.
Allele discrimination was performed through allele-specific
hybridization. In total, 248,535 SNPs, distributed as evenly as
possible throughout the genome, were genotyped on the 382 QFP trios
for a total of 372,802,500 genotypes. These markers were mostly
selected from various databases including the .about.1.6 million
SNP database of Perlegen Life Sciences (Patil, 2001); several
thousand were obtained from the HapMap consortium database and/or
dbSNP at NCBI. The SNPs were chosen to maximize uniformity of
genetic coverage and to cover a distribution of allele frequencies.
All SNPs that did not pass the quality controls for the assay, that
is, that had a minor allele frequency of less than 1%, a Mendelian
error rate within trios greater than 1%, that deviated
significantly from the Hardy-Weinberg equilibrium, or that had
excessive missing data (cut-off at 1% missing values or higher)
were removed from the analysis.
[0221] Genetic analysis was performed on a total of 165,803 SNPs
(158,811 autosomal, 6851 X chromosome and 141 Y chromosome). The
average gap size was approximately 17 kb. Of the 165,803 markers,
.about.140,000 had a minor allele frequency (MAF) greater than 10%
for the QFP.
[0222] The genotyping information was entered into a Unified
Genotype Database (a proprietary database under development) from
which it was accessed using custom-built programs for export to the
genetic analysis pipeline. Analyses of these genotypes were
performed with the statistical tools described in Example 3. The
GWS permitted the identification of 371 candidate chromosomal
regions linked to Crohn's disease (Table 1).
Example 3
Genetic Analysis
[0223] 1. Dataset Quality Assessment
[0224] Prior to performing any analysis, the dataset from the GWS
was verified for completeness of the trios. The programs Famcheck
and Fampull removed any trios with abnormal family structure or
missing individuals (e.g. trios without a proband, duos,
singletons, etc.), and calculated the total number of complete
trios in the dataset. The trios were also tested to make sure that
no subjects within the cohort were related more closely than second
cousins (6 meiotic steps).
[0225] Subsequently, the program DataStats was used to calculate
the following statistics per marker and per family: [0226] Minor
allele frequency (MAF) for each marker; Missing values for each
marker and family; Hardy Weinberg Equilibrium for each marker; and
[0227] Mendelian segregation error rate.
[0228] The following acceptance criteria were applied for internal
analysis purposes: [0229] MAF>1%; [0230] Missing values <1%;
[0231] Observed non-Mendelian segregation <0.33%; [0232] Non
significant deviation in allele frequencies from Hardy Weinberg
equilibrium.
[0233] Markers or families not meeting these criteria were removed
from the dataset in the following step. Analyses of variance were
performed using the algorithm GenAnova, to assess whether families
or markers have a greater effect on missing values and/or
non-Mendelian segregation. This was used to determine the smallest
number of data points to remove from the dataset in order to meet
the requirements for missing values and non-Mendelian segregation.
The families and/or markers were removed from the dataset using the
program DataPull, which generates an output file that is used for
subsequent analysis of the genotype data.
[0234] 2. Phase Determination
[0235] The program PhaseFinderSNP2.0 was used to determine phase
from trio data on a marker-by-marker, trio-by-trio basis. The
output file contains haplotype data for all trio members, with
ambiguities present when all trio members are heterozygous or where
data is missing. The program AlIHaps2 PatCtrl was then used to
determine case and control haplotypes and to prepare the data in
the proper input format for the next stage of analysis, using the
expectation maximization algorithm, PL-EM, to call phase on the
remaining ambiguities. This stage consists of several modules for
resolution of the remaining phase ambiguities. PLEMInOut1 was first
used to recode the haplotypes for input into the PL-EM algorithm in
15-marker blocks for the genome wide scan data and for 11 marker
blocks for fine and ultra-fine mapping data sets. The haplotype
information was encoded as genotypes, allowing for the entry of
known phase into the algorithm; this method limits the possible
number of estimated haplotypes conditioned on already known phase
assignments. The PL-EM algorithm was used to estimate haplotypes
from the "pseudo-genotype" data in 11 or 15-marker windows,
advancing in increments of one marker across the chromosome. The
results were then converted into multiple haplotype files using the
program PLEMInOut2. Subsequently PLEMBlockGroup was used to convert
the individual 11 or 15-marker block files into one continuous
block of haplotypes for the entire chromosome and to generate files
for further analysis by LDSTATS and SINGLETYPE. PLEMBlockGroup
takes the consensus estimation of the allele call at each marker
over all separate estimations (most markers are estimated 11-15
different times as the 11 or 15 marker blocks pass over their
position).
[0236] 3. Haplotype Association Analysis
[0237] Haplotype association analysis was performed using the
program LDSTATS. LDSTATS tests for association of haplotypes with
the disease phenotype. The algorithms LDSTATS (v2.0) and LDSTATS
(v4.0) define haplotypes using multi-marker windows that advance
across the marker map in one-marker increments. Windows can contain
any odd number of markers specified as a parameter of the
algorithm. Other marker windows can also be used. At each position
the frequency of haplotypes in cases and controls was calculated
and a chi-square statistic was calculated from case control
frequency tables. For LDSTATS v2.0, the significance of the
chi-square for single marker and 3-marker windows was calculated as
Pearson's chi-square with degrees of freedom. Larger windows of
multi-allelic haplotype association were tested using Smith's
normalization of the square root of Pearson's Chi-square. In
addition, LDSTATS v2.0 calculates Chi-square values for the
transmission disequilibrium test (TDT) for single markers in
situations where the trios consisted of parents and an affected
child.
[0238] LDSTATS v4.0 calculates significance of chi-square values
using a permutation test in which case-control status is randomly
permuted until 350 permuted chi-square values are observed that are
greater than or equal to chi-square value of the actual data. The P
value is then calculated as 350/the number of permutations
required.
[0239] The best signal at a given location was determined by
comparing the significance (p-value) of the association with Crohn
disease for window sizes of 1, 3, 5, 7, and 9 SNPs, and selecting
the most significant window. For a given window size at a given
location, the association with Crohn disease was evaluated by
comparing the overall distribution of haplotypes in the cases with
the overall distribution of haplotypes in the controls. Haplotypes
with a relative risk greater than one increase the risk of
developing Crohn disease while haplotypes with a relative risk less
than one are protective and decrease the risk.
[0240] 4. Singletype Analysis
[0241] The SINGLETYPE algorithm assesses the significance of
case-control association for single markers using the genotype data
from the laboratory as input in contrast to LDSTATS single marker
window analyses, in which case-control alleles for single markers
from estimated haplotypes in file, hapatctr.txt, as input.
SINGLETYPE calculates P values for association for both alleles, 1
and 2, as well as for genotypes, 11, 12, and 22, and plots these as
-log.sub.10 P values for significance of association against marker
position.
[0242] 5. Conditional Haplotype Analyses
[0243] Conditional haplotype analyses were performed on subsets of
the original set of 382 cases and 382 controls using the program
LDSTATS (v2.0). The selection of a subset of cases and their
matched controls was based on the carrier status of cases at a gene
or locus of interest. We selected the gene NOD2 (alias CARD15) on
chromosome 16 and the gene IL23R on chromosome 1 based on our
association findings using LDSTAT (v2.0) in our fine mapping of
these two loci with 477 trios (see below). Additionally, we
selected the gene IRAK2 on chromosome 3 and identified haplotypes
based on SNPs in the genome wide scan on 382 trios. The most
significant association signal in NOD2, using build 35, was
obtained with a haplotype window of size 7 containing SNPs
corresponding to SEQ IDs 21467, 21468, 21469, 21470, 21471, 21472,
21473 (see Table below for conversion to the specific DNA alleles
used). A reduced haplotype diversity was observed and we selected
two sets of risk haplotypes for conditional analyses. The first set
consisted of haplotypes 2121222 and 1121211 and the second set
contained the above two haplotypes and haplotype 2121211. Using the
first set, we partitioned the cases into two groups; the first
group consisting of those cases that were carrier of a risk
haplotype and the second group consisting of the remaining cases,
the non-carriers. The resulting sample sizes were respectively 125
and 227. The sample sizes were 200 and 152 for the two groups when
we partitioned the cases into two groups using the second set
containing three risk haplotypes. LDSTAT (v2.0) was run in each
group and regions showing association with Crohn's disease are
reported in Tables 5-20. Regions associated with Crohn's disease in
the group of carriers (B35#has_NOD2_cr.sub.--3haps) indicate the
presence of an epistatic interaction between risk factors in those
regions and risk factors in NOD2 (Table 5). Regions associated with
Crohn's disease in the group of non carriers
(B35#not_NOD2_cr.sub.--2haps) indicate the existence of risk
factors acting independently of NOD2 (Table 6). The most
significant association signal in NOD2, using build 36, was
obtained with a haplotype window of size 7 containing SNPs
corresponding to SEQ IDs 21474, 21467, 21468, 21469, 21470, 21471,
21472 (see Table below for conversion to the specific DNA alleles
used). A reduced haplotype diversity was observed and we selected a
set of risk haplotypes and a set of protective haplotypes for
conditional analyses. The risk set consisted of haplotypes 1112121
and 1212122, while excluding genotype 1212122/1221121 due to
dominance effects. The protective set consisted of allele 1222211,
excluding herozygote carriers of either allele 1112121 or 1212122,
and of homozygote individuals with genotype 1221121/1221121. The
resulting sample sizes were 132 and 238 for the subsetting based on
the risk set and 158 and 212 for the protective set, respectively
for the carriers and the non-carriers. LDSTAT (v2.0) was run in
each group and regions showing association with Crohn's disease are
reported in Tables 5-20. Regions associated with Crohn's disease in
the group of non-carriers of a risk haplotype (B36#not_NOD2_cr) and
in the group of carriers of a protective haplotype
(B36#has_NOD2_cp) indicate the existence of risk factors acting
independently of NOD2 (Tables 20 and 17).
[0244] A second conditional analysis was performed using the gene
IL23R on chromosome 1. The most significant association in IL23R,
using build 35, was obtained with a haplotype window of size 9
containing SNPs corresponding to SEQ IDs 21481, 21482, 21483,
21484, 21485, 21486, 21487, 21488, 21489 (see Table below for
conversion to the specific DNA alleles used). A reduced haplotype
diversity was observed and we selected one set of protective
haplotypes and a risk haplotype for conditional analyses. The
protective set consisted of haplotypes 212111122 and 212111121 and
the risk haplotype was 221211121. However, due to dominance effects
involving the risk haplotype, we also considered the risk haplotype
221211121 while excluding heterozygotes involving haplotypes
122122212, 222122211 or 212111122 and the risk haplotype. Using the
set of protective haplotypes, we partitioned the cases into two
groups; the first group consisting of those cases that were carrier
of a protective haplotype and the second group consisting of the
remaining cases, the non-carriers. The resulting sample sizes were
respectively 204 and 162. LDSTATS (v2.0) was run in each group and
regions showing association with Crohn's disease are reported in
Tables 5-20. Regions associated with Crohn's disease in the group
of carriers (B35#has_IL23R_cp2hap), indicate the existence of risk
factors acting independently of IL23R. Regions associated with
Crohn's disease in the group of non-carriers (B35#not_IL23R_cp2hap)
indicate the presence of an epistatic interaction between risk
factors in those regions and risk factors in IL23R (Tables 7 and
10). We repeated the process of partitioning the cases into two
groups using the risk haplotype in IL23R. The carriers were cases
with a haplotype in the risk set, the non-carriers were the
remaining set of cases. The sample sizes for the two groups were
184 and 182 respectively. Regions associated with Crohn's disease
in the group of carriers (B35#has_IL23R_cr) and
(B35#has_IL23R_cr_not3) indicate the presence of an epistatic
interaction between risk factors in those regions and risk factors
in IL23R (Tables 8 and 9). Regions associated with Crohn's disease
in the group of non-carriers (B35#not_IL23R_cr) and
(B35#not_IL23R_cr_not3) indicate the presence of risk factors
acting independently of IL23R (Tables 11 and 12). The most
significant association in IL23R, using build 36, was obtained with
a haplotype window of size 5 containing SNPs corresponding to SEQ
IDs 21488, 21489, 21492, 21493, 21494 (see Table below for
conversion to the specific DNA alleles used). A reduced haplotype
diversity was observed and we selected one set of risk haplotypes
for conditional analyses. The risk set consisted of haplotype
21222, while excluding heterozygote genotypes with haplotypes 12122
or 22122 and haplotype 21222 due to dominance effects. We
partitioned the cases into two groups; the first group consisting
of those cases that were carrier of a haplotype in the risk set and
the second group consisting of the remaining cases, the
non-carriers. The resulting sample sizes were respectively 194 and
174. LDSTATS (v2.0) was run in each group and regions showing
association with Crohn's disease are reported in Tables 5-20.
Regions associated with Crohn's disease in the group of carriers
(B36#has_IL23R_cr) indicate the presence of an epistatic
interaction between risk factors in those regions and risk factors
in IL23R (Table 15). Regions associated with Crohn's disease in the
group of non-carriers of a risk haplotype (B36#not_IL23R_cr)
indicate the existence of risk factors acting independently of
IL23R (Table 16). When considering the program LDSTATS (v4.0), the
most significant association in IL23R, using build 36, was obtained
with a haplotype window of size 3 containing SNPs corresponding to
SEQ IDs 21482, 21483, 21484 (see Table below for conversion to the
specific DNA alleles used). A reduced haplotype diversity was
observed and we selected one set of risk and a set of protective
haplotypes for conditional analyses. The risk set consisted of
genotypes 212/221 and 212/212. We partitioned the cases into two
groups; the first group consisting of those cases that were carrier
of a genotype in the risk set and the second group consisting of
the remaining cases, the non-carriers. The resulting sample sizes
were respectively 112 and 256. The protective set consisted of
genotypes 121/221 and 121/121. As was done with the risk set, we
partitioned the cases into two groups; the first group consisting
of those cases that were carrier of a genotype in the protective
set and the second group consisting of the remaining cases, the
non-carriers. The resulting sample sizes were respectively 111 and
257. LDSTATS (v2.0) was run in each group and regions showing
association with Crohn's disease are reported in Tables 5-20.
Regions associated with Crohn's disease in the group of carriers
(B36#has_IL23R-1_cr) indicate the presence of an epistatic
interaction between risk factors in those regions and risk factors
in IL23R (Table 14). Regions, associated with Crohn's disease in
the group of non-carriers of a risk haplotype (B36#not_IL23R-1cr)
and in the group of carriers of a protective haplotype
(B36#has_IL23R-1_cp) indicate the existence of risk factors acting
independently of IL23R (Tables 18 and 13).
[0245] A third conditional analysis was performed using the gene
IRAK2 on chromosome 3. The most significant association in IRAK2,
using build 36, was obtained with a single SNP corresponding to SEQ
ID 21498 (see Table below for conversion to the specific DNA
alleles used). A reduced haplotype diversity was observed and we
selected a risk genotype for conditional analyses. The risk set
consisted of genotype 2/2. We partitioned the cases into two
groups; the first group consisting of those cases that were carrier
of the risk genotype and the second group consisting of the
remaining cases, the non-carriers. The resulting sample sizes were
respectively 111 and 259. LDSTATS (v2.0) was run in each group and
regions showing association with Crohn's disease are reported in
Table 5-20. Regions, associated with Crohn's disease in the group
of non carriers of a risk haplotype (B36#not_IRAK2_cr) indicate the
existence of risk factors acting independently of IRAK2 (Table
19).
[0246] For each region that was associated with Crohn's disease in
the conditional analyses, we report in Tables 21-36 the allele
frequencies and the relative risk (RR) for the haplotypes
contributing to the best signal at each SNP in the region. The best
signal at a given location was determined by comparing the
significance (p-value) of the association with Crohn's disease for
window sizes of 1, 3, 5, 7, and 9 SNPs, and selecting the most
significant window. For a given window size at a given location,
the association with Crohn's disease was evaluated by comparing the
overall distribution of haplotypes in the cases with the overall
distribution of haplotypes in the controls. Haplotypes with a
relative risk greater than one increase the risk of developing
Crohn's disease while haplotypes with a relative risk less than one
are protective and decrease the risk.
TABLE-US-00001 DNA alleles used in haplotypes (NOD2/CARD15 Build
35) SeqID 21475 21476 21477 21478 21479 21480 21480 Position B35
49303427 49305205 49308676 49308899 49310925 49314041 49314275
Allele T/C T/C T/G A/G A/G G/C T/C 2121222 C T G A G C C 1121211 T
T G A G G T 2121211 C T G A G G T DNA alleles used in haplotypes
(IL23R Build 35) SeqID 21481 21482 21483 21484 21485 21486 21487
21490 21491 Position B35 67381537 67382937 67384786 67387749
67388943 67390943 67392949 67395507 67397137 Alleles T|G A|G A|G
T|G T|C A|C T|C T|C T|C 221211121 G G A G T A T C T 122122212 T G G
T C C C T C 222122211 G G G T C C C T T 212111122 G A G T T A T C C
212111121 G A G T T A T C T DNA alleles used in haplotypes
(NOD2/CARD15 Build 36) SeqID 21474 21475 21476 21477 21478 21479
21480 Position B36 49302189 49303427 49305205 49308676 49308899
49310925 49314041 Alleles A|G T|C T|C T|G A|G A|G G|C 1112121 A T T
G A G G 1212122 A C T G A G C 1221121 A C C T A G G 1222211 A C C G
G A G DNA alleles used in haplotypes (IL23R Build 36) SeqID 21490
21491 21492 21493 21494 Position B36 67456074 67457704 67457975
67458031 67459652 Alleles T|C T|C T|C A|G T|C 12122 T C T G C 21222
C T C G C 22122 C C T G C DNA alleles used in haplotypes (iL23R
Build 36 LDV4) SeqID 21495 21496 21497 Position B36 67443504
67445353 67448316 Alleles A|G A|G T|G 121 A G T 212 G A G 221 G G T
DNA alleles used in haplotypes (IRAK2 Build 36) SeqID 21498
Position B36 10253980 Alleles A|C 1 A 2 C
Example 4
Gene Identification and Characterization
[0247] A series of gene characterization was performed for each
candidate region described in Table 1. Any gene or EST mapping to
the interval based on public map data or proprietary map data was
considered as a candidate Crohn's disease gene. The approach used
to identify all genes located in the critical regions is described
below.
[0248] Public Gene Mining
[0249] Once regions were identified using the analyses described
above, a series of public data mining efforts were undertaken, with
the aim of identifying all genes located within the critical
intervals as well as their respective structural elements (i.e.,
promoters and other regulatory elements, UTRs, exons and splice
sites). The initial analysis relied on annotation information
stored in public databases (e.g. NCBI, UCSC Genome Bioinformatics,
Entrez Human Genome Browser, OMIM--see below for database URL
information). Tables 2-4 lists the genes that have been mapped to
the 654 candidate regions.
TABLE-US-00002 Database URLs Name URL Biocarta
http://www.biocarta.com/ BioCyc http://www.biocyc.org/ Biomolecular
Interaction Network http://bind.ca/ Database (BIND) Database of
Interacting Proteins http://dip.doe-mbi.ucla.edu/ Gene Expression
Omnibus http://www.ncbi.nlm.nih.gov/geo/ Human Genome Browser
http://www.ensembl.org/Homo_sapiens/ Interdom
http://interdom.lit.org.sg/help/term.php Kyoto Encyclopedia of
Genes and http://www.genome.jp/kegg/ Genomes (KEGG) Molecular
Interactions Database http://mint.bio.uniroma2.it/mint/ (MINT)
National Center for Biotechnology http://www.ncbi.nlm.nih.gov/
Information (NCBI) Online Mendelian Inheritance in
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM Man (OMIM)
OmniViz http://www.omniviz.com/applications/omni_viz.htm Pathway
Enterprise http://www.omniviz.com/applications/pathways.htm
Reactome http://www.reactome.org/ Transpath
http://www.biobase.de/pages/products/transpath.html UCSC Genome
Bioinformatics http://genome.ucsc.edu/index.html?org=Human UniGene
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene
[0250] For some genes the available public annotation was
extensive, whereas for others very little was known about a gene's
function. Customized analysis was therefore performed to
characterize genes that corresponded to this latter class.
Importantly, the presence of rare splice variants and artifactual
ESTs was carefully evaluated. Subsequent cluster analysis of novel
ESTs provided an indication of additional gene content in some
cases. The resulting clusters were graphically displayed against
the genomic sequence, providing indications of separate clusters
that may contribute to the same gene, thereby facilitating
development of confirmatory experiments in the laboratory. While
much of this information was available in the public domain, the
customized analysis performed revealed additional information not
immediately apparent from the public genome browsers.
[0251] A unique consensus sequence was constructed for each splice
variant and a trained reviewer assessed each alignment. This
assessment included examination of all putative splice junctions
for consensus splice donor/acceptor sequences, putative start
codons, consensus Kozak sequences and upstream in-frame stops, and
the location of polyadenylation signals. In addition, conserved
noncoding sequences (CNSs) that could potentially be involved in
regulatory functions were included as important information for
each gene. The genomic reference and exon sequences were then
archived for future reference. A master assembly that included all
splice variants, exons and the genomic structure was used in
subsequent analyses (i.e., analysis of polymorphisms). Table 3
lists gene clusters based on the publicly available EST and cDNA
clustering algorithm, ECGene.
[0252] An important component of these efforts was the ability to
visualize and store the results of the data mining efforts. A
customized version of the highly versatile genome browser GBrowse
(http://www.gmod.org/) was implemented in order to permit the
visualization of several types of information against the
corresponding genomic sequence. In addition, the results of the
statistical analyses were plotted against the genomic interval,
thereby greatly facilitating focused analysis of gene content.
[0253] Computational Analysis of Genes and GeneMaps
[0254] In order to assist in the prioritization of candidate genes
for which minimal annotation existed, a series of computational
analyses were performed that included basic BLAST searches and
alignments to identify related genes. In some cases this provided
an indication of potential function. In addition, protein domains
and motifs were identified that further assisted in the
understanding of potential function, as well as predicted cellular
localization.
[0255] A comprehensive review of the public literature was also
performed in order to facilitate identification of information
regarding the potential role of candidate genes in the
pathophysiology of Crohn's disease. In addition to the standard
review of the literature, public resources (Medline and other
online databases) were also mined for information regarding the
involvement of candidate genes in specific signaling pathways. A
variety of pathway and yeast two hybrid databases were mined for
information regarding protein-protein interactions. These included
BIND, MINT, DIP, Interdom, and Reactome, among others. By
identifying homologues of genes in the Crohn's candidate regions
and exploring whether interacting proteins had been identified
already, knowledge regarding the GeneMaps for Crohn's disease was
advanced. The pathway information gained from the use of these
resources was also integrated with the literature review efforts,
as described above.
[0256] Genes identified in the WGAS were evaluated using the
Ingenuity Pathway Analysis application (IPA, Ingenuity systems) in
order to identify direct biological interactions between these
genes, and also to identify molecular regulators acting on those
genes (indirect interactions) that could be also involved in CD.
The purpose of this effort was to decipher the molecules involved
in contributing to CD. These gene interaction networks are very
valuable tools in the sense that they facilitate extension of the
map of gene products that could represent potential drug targets
for CD.
[0257] Expression Studies
[0258] In order to determine the expression patterns for genes,
relevant information was first extracted from public databases. The
UniGene database, for example, contains information regarding the
tissue source for ESTs and cDNAs contributing to individual
clusters. This information was extracted and summarized to provide
an indication in which tissues the gene was expressed. Particular
emphasis was placed on annotating the tissue source for bona fide
ESTs, since many ESTs mapped to Unigene clusters are artifactual.
In addition, SAGE and microarray data, also curated at NCBI (Gene
Expression Omnibus), provided information on expression profiles
for individual genes. Particular emphasis was placed on identifying
genes that were expressed in tissues known to be involved in the
pathophysiology of Crohn's disease, e.g. intestinal and immune
system tissues.
[0259] Polymorphism Analysis
[0260] Polymorphisms identified in candidate genes, including those
from the public domain as well as those identified by sequencing
candidate genes and regions, are evaluated for potential function.
Initially, polymorphisms are examined for potential impact upon
encoded proteins. If the protein is a member of a gene family with
reported 3-dimensional structural information, this information is
used to predict the location of the polymorphism with respect to
protein structure. This information provided insight into the
potential role of polymorphisms in altering protein or ligand
interactions, as well as suitability as a drug target. In a second
phase of analysis we evaluate the potential role of polymorphisms
in other biological phenomena, including regulation of
transcription, splicing and mRNA stability, etc. There are many
examples of the functional involvement of naturally occurring
polymorphisms in these processes. As part of this analysis,
polymorphisms located in promoter or other regulatory elements,
canonical splice sites, exonic and intronic splice enhancers and
repressors, conserved noncoding sequences and UTRs are
localized.
Example 5
SNP and Polymorphism Discovery (SNPD)
[0261] Candidate regions are selected for sequencing in order to
identify all polymorphisms. In cases where the critical interval,
identified by fine mapping, is relatively small (.about.50 kb), the
entire region, including all introns, is sequenced to identify
polymorphisms. In situations where the region is large (>50 kb),
candidate genes are prioritized for sequencing, and/or only
functional gene elements (promoters, exons and splice sites) are
sequenced.
[0262] The samples sequenced are selected according to which
haplotypes contribute to the association signal observed in the
region. The purpose is to select a set of samples that covered all
the major haplotypes in the given region. Each major haplotype must
be present in a few copies. The first step therefore consisted of
determining the major haplotypes in the region to be sequenced.
[0263] Once a region is defined with the two boundary markers, all
the markers used in fine mapping that are located within the region
are used to determine the major haplotypes. Long haplotypes
covering the whole region are thus inferred using the middle marker
as an anchor. The results included two series of haplotype themes
that define the major haplotypes, comparing the cases and the
controls. This exercise is repeated using an anchor in the
peripheral regions to ensure that major haplotype subsets that are
not anchored at the original middle marker are not missed.
[0264] Once the major haplotypes are determined as described above,
appropriate genomic DNA samples are selected such that each major
haplotype and haplotype subset were represented in at least two to
four copies.
[0265] The sequencing protocol included the following steps, once a
region was delimited:
[0266] 1. Primer Design
[0267] The design of the primers is performed using a proprietary
primer design tool. A primer quality control step was included in
the primer design process. Primers that successfully passed the
control quality process are synthesized by Integrated DNA
Technologies (IDT). The sense and anti-sense oligos are separated
such that the sense oligos were placed on one plate in the same
position as their anti-sense counterparts on another plate. Two
additional plates are created from each storage plate, one for use
in PCR and the other for sequencing. For PCR, the sense and
anti-sense oligos of the same pair are combined in the same well to
achieve a final concentration of 1.5 .mu.M for each
oligonucleotide.
[0268] 2. PCR Optimization
[0269] PCR conditions are optimized by testing a variety of
conditions that included varying salt concentrations and
temperatures, as well as including various additives. PCR products
are checked for robust amplification and minimal background by
agarose gel electrophoresis.
[0270] 3. PCR on Selected Samples
[0271] PCR products used for sequencing are amplified using the
conditions chosen during optimization. The PCR products are
purified free of salts, dNTPs and unincorporated primers by use of
a MultiScreen PCR384 filter plate manufactured by Millipore.
Following PCR, the amplicons are quantified by use of a lambda/Hind
III standard curve. This is done to ensure that the quantity of PCR
product required for sequencing had been generated. The raw data is
measured against the standard curve data in Excel by use of a
macro.
[0272] 4. Sequencing
[0273] Sequencing of PCR products is performed by DNA Landmarks
using ABI 3730 capillary sequencing instruments.
[0274] 5. Sequence Analysis
[0275] The ABI Prism SeqScape software (Applied Biosystems) is used
for SNP identification. The chromatogram trace files are imported
into a SeqScape sequencing project and the base calling is
automatically performed. Sequences are then aligned and compared to
each other using the SeqScape program. The base calling is checked
manually, base by base; editing was performed if needed.
Example 6
Ultra Fine Mapping (UFM)
[0276] Once polymorphisms are identified by sequencing efforts as
described in Example 5, additional genotyping of all newly found
polymorphisms is performed on the samples used in the fine mapping
study. Various types of genotyping assays may need to be utilized
based on the type of polymorphism identified (i.e., SNP, indel,
microsatellite). The assay type can be, but is not restricted to,
Sentrix Assay Matrix on Illumina BeadStations, microsatellite on
MegaBACE, SNP on ABI or Orchid. The frequencies of genotypes and
haplotypes in cases and controls are analyzed in a similar manner
as the GWS data. By examining all SNPs in a region, polymorphisms
are identified that increase an individual's susceptibility to
Crohn's disease. The result of ultra-fine mapping is to identify
the polymorphism that is most associated with disease phenotype as
part of the search for the actual DNA polymorphism that confers
susceptibility to disease.
[0277] All publications, patents and patent applications mentioned
in the specification and reference list are herein incorporated by
reference in their entirety for all purposes. Various modifications
and variations of the described method and system of the invention
will be apparent to those skilled in the art without departing from
the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
that are obvious to those skilled in molecular biology, genetics,
or related fields are intended to be within the scope of the
following claims.
[0278] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); DNA Cloning, Volumes I and H (D. N. Glover ed., 4);
Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al.
U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames
& S. J. Higgins eds. 1984); Transcription And Translation (B.
D. Haines & S. J. Higgins eds. 1984); Culture Of Animal Cells
(R. 1. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And
Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the treatise, Methods In Enzymology
(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian
Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
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5,173,414
TABLE-US-00003 [0691] Lengthy table referenced here
US20090181380A1-20090716-T00001 Please refer to the end of the
specification for access instructions.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090181380A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090181380A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090181380A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References